Invertebrate MicroRNAs

ABSTRACT

This invention provides plants having resistance to invertebrate pests. More specifically, this invention discloses a non-natural transgenic plant cell expressing at least one invertebrate miRNA in planta for suppression of a target gene of an invertebrate pest or of a symbiont associated with the invertebrate pest. Also provided are recombinant DNA constructs for expression of at least one invertebrate miRNA in planta, a non-natural transgenic plant containing the non-natural transgenic plant cell of this invention, a non-natural transgenic plant grown from the non-natural transgenic plant cell of this invention, and non-natural transgenic seed produced by the non-natural transgenic plants, as well as commodity products produced from a non-natural transgenic plant cell, plant, or seed of this invention. This invention further provides a method of suppressing at least one target gene of an invertebrate pest of a plant or of a symbiont associated with the invertebrate, including providing a plant including the non-natural transgenic plant cell of this invention, wherein the invertebrate is the invertebrate pest, the recombinant DNA is transcribed in the non-natural transgenic plant cell to the recombinant miRNA precursor, and when the invertebrate pest ingests the recombinant miRNA precursor, the at least one target gene is suppressed.

CROSS-REFERENCE TO RELATED APPLICATIONS AND INCORPORATION OF SEQUENCELISTINGS

This application claims the benefit of priority of U.S. ProvisionalPatent Application 60/890,705, filed 20 Feb. 2007, which is incorporatedby reference in its entirety herein. The sequence listing contained inthe file named “38-21_(—)54478_A.txt”, which is 35 kilobytes (measuredin operating system MS-Windows) and was created on 15 Feb. 2007 andfiled with U.S. Provisional Patent Application 60/890,705 on 20 Feb.2007, is incorporated herein by reference.

INCORPORATION OF SEQUENCE LISTING

The sequence listing that is contained in the file named“38-21_(—)54478_B.txt”, which is 35 kilobytes (measured in operatingsystem MS-Windows), created on 12 Feb. 2008, is filed herewith andincorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to novel microRNAs and microRNA precursorsidentified from invertebrates, as well as recombinant DNA constructsincluding such novel miRNAs, miRNA precursors, and miRNA recognitionsites corresponding to the miRNAs. Also disclosed are non-naturaltransgenic plant cells, plants, and seeds containing in their genome arecombinant DNA construct of this invention. Further provided aremethods of gene suppression using recombinant DNA constructs of thisinvention.

BACKGROUND OF THE INVENTION

MicroRNAs (miRNAs) are non-protein coding RNAs, generally of betweenabout 19 to about 25 nucleotides (commonly about 20-24 nucleotides inplants), that guide cleavage in trans of target transcripts, negativelyregulating the expression of genes involved in various regulation anddevelopment pathways (Bartel (2004) Cell, 116:281-297). In some cases,miRNAs serve to guide in-phase processing of siRNA primary transcripts(see Allen et al. (2005) Cell, 121:207-221).

Many microRNA genes (MIR genes) have been identified and made publiclyavailable in a database (‘miRBase”, available on line atmicroma.sanger.ac.uk/sequences; also see Griffiths-Jones et al. (2003)Nucleic Acids Res., 31 :439-441). MicroRNAs were first reported fromnematodes and have since been identified in other invertebrates; see,for example, Lee and Ambros (2001) Science, 294:862-864; Lim et al.(2003) Genes Dev., 17:991-1008; Stark et al. (2007) Genome Res.,17:1865-1879. MIR genes have been reported to occur in intergenicregions, both isolated and in clusters in the genome, but can also belocated entirely or partially within introns of other genes (bothprotein-coding and non-protein-coding). For a recent review of miRNAbiogenesis, see Kim (2005) Nature Rev. Mol Cell Biol., 6:376-385.Transcription of MIR genes can be, at least in some cases, underpromotional control of a MIR gene's own promoter. The primarytranscript, termed a “pri-miRNA”, can be quite large (several kilobases)and can be polycistronic, containing one or more pre-miRNAs (fold-backstructures containing a stem-loop arrangement that is processed to themature miRNA) as well as the usual 5′ “cap” and polyadenylated tail ofan mRNA. See, for example, FIG. 1 in Kim (2005) Nature Rev. Mol. CellBiol., 6:376-385.

Maturation of a mature miRNA from its corresponding precursors(pri-miRNAs and pre-miRNAs) differs significantly between animals andplants. For example, in plant cells, microRNA precursor molecules arebelieved to be largely processed to the mature miRNA entirely in thenucleus, whereas in animal cells, the pri-miRNA transcript is processedin the nucleus by the animal-specific enzyme Drosha, followed by exportof the pre-miRNA to the cytoplasm where it is further processed to themature miRNA. Mature miRNAs in plants are typically 21 nucleotides inlength, whereas in animals 22 nucleotide long miRNAs are most commonlyfound. For a recent review of miRNA biogenesis in both plants andanimals, see Kim (2005) Nature Rev. Mol. Cell Biol., 6:376-385.Additional reviews on miRNA biogenesis and function are found, forexample, in Bartel (2004) Cell, 116:281-297; Murchison and Hannon (2004)Curr. Opin. Cell Biol., 16:223-229; and Dugas and Bartel (2004) Curr.Opin. Plant Biol., 7:512-520. Furthermore, although one recent reportdescribes a miRNA (miR854) from Arabidopsis that also is found inanimals (Arteaga-Vazquez et al. (2006) Plant Cell, 18:3355-3369), miRNAconservation generally appears to be kingdom-specific. Animal miRNAshave many characteristic dissimilar to their plant counterparts,including shorter miRNA precursor fold-backs (about 90 nucleotides inanimals versus about 180 nucleotides in plants) with the mature miRNAsequence tending to be found at the base of the stem, a higher number ofmismatches within the foldback, and deriviation from from polycistronicmessages. Whereas animal miRNAs generally anneal imperfectly to the 3′untranslated region (UTR) of their target mRNA, most plant miRNAs arecharacterized by having perfect or near-perfect complementarity to theirtarget sequence, which is usually in the coding region, with only a fewexamples of miRNAs having binding sites within the UTRs of the targetmRNA; see Rhoades et al. (2002) Cell, 110:513-520; Jones-Rhoades et al.(2006) Annu. Rev. Plant Biol., 57:19-53. These significant differencesbetween plant and animal miRNAs make it generally unlikely that miRNAswill be processed and function across kingdoms.

Transgenic expression of miRNAs (whether a naturally occurring sequenceor an artificial sequence) can be employed to regulate expression of themiRNA's target gene or genes. Inclusion of a miRNA recognition site in atransgenically expressed transcript is also useful in regulatingexpression of the transcript; see, for example, Parizotto et al. (2004)Genes Dev., 18:2237-2242. Recognition sites of miRNAs have beenvalidated in all regions of an mRNA, including the 5′ untranslatedregion, coding region, and 3′ untranslated region, indicating that theposition of the miRNA target site relative to the coding sequence maynot necessarily affect suppression (see, e.g., Jones-Rhoades and Bartel(2004). Mol. Cell, 14:787-799, Rhoades et al. (2002) Cell, 110:513-520,Allen et al. (2004) Nat. Genet., 36:1282-1290, Sunkar and Zhu (2004)Plant Cell, 16:2001-2019). Because miRNAs are important regulatoryelements in eukaryotes, transgenic suppression of miRNAs is useful formanipulating biological pathways and responses. Finally, promoters ofMIR genes can have very specific expression patterns (e.g.,cell-specific, tissue-specific, temporally specific, or inducible), andthus are useful in recombinant constructs to induce such specifictranscription of a DNA sequence to which they are operably linked.Various utilities of miRNAs, their precursors, their recognition sites,and their promoters are described in detail in U. S. Patent ApplicationPublication 2006/0200878 A1, incorporated by reference herein.Non-limiting examples of these utilities include: (1) the expression ofa native miRNA or miRNA precursor sequence to suppress a target gene;(2) the expression of an engineered (non-native) miRNA or miRNAprecursor sequence to suppress a target gene; (3) expression of atransgene with a miRNA recognition site, wherein the transgene issuppressed when the mature miRNA is expressed; (4) expression of atransgene driven by a miRNA promoter.

Animal miRNAs have been utilized as precursors to express specificmiRNAs in animal cells; for example, the human miR-30 precursor wasexpressed as the native sequence and as a modified (artificial orengineered) miRNA in cultured cells (Zeng et al. (2002) Mol. Cell,9:1327-1333, and Zeng et al. (2005) J. Biol. Chem., 280:27595-27603). Asingle mature miRNA is precisely processed from a given precursor, andtherefore such “artificial” or engineered miRNAs offer an advantage overdouble-stranded RNA (dsRNA) in that only a specific miRNA sequence isexpressed, limiting potential off-target effects. Although animal miRNAstypically interact with imperfect target sequences in the 3′ UTR,synthetic miRNAs with perfect target complementarity also can guidetarget cleavage (see Zeng et al. (2003) RNA, 9:112-123 and Zeng et al(2003) Proc. Natl. Acad. Sci. U.S.A., 100:9779-9784).

Small RNAs, referred to as short interfering RNAs (siRNAs) and microRNAs (miRNAs), have been shown to regulate gene expression in plants andanimals (Valencia-Sanchez et al. (2006) Genes Dev., 20:515-524; Nelsonet al. (2003) Trends Biochem. Sci., 28:534-540). Experimental alterationof siRNA levels result in phenotypic effects in nematodes (Timmons andFire (1998) Nature, 395:8543). A plant that transgenically expressedsiRNA complementary to the root-knot nematode 16D10 gene was shown tohave resistance to four species of root-knot nematodes (Huang et al.(2006) Proc. Natl. Acad. Sci. U.S.A., 103:14302-14306). This inventiondiscloses the use of recombinant invertebrate miRNAs expressed in plantato similarly regulate expression in an invertebrate that ingests themiRNAs.

This invention discloses recombinant DNA constructs encodinginvertebrate mature miRNAs and their miRNA precursors, which aredesigned to be expressed in planta. In some embodiments, theinvertebrate miRNA precursors are engineered to express artificialmiRNAs designed to suppress or silence specific invertebrate genes andthereby confer upon a plant expressing the miRNAs resistance to aninvertebrate that ingests the miRNAs. In many cases, RNAi (siRNA ormiRNA) transcripts that are intended to suppress an invertebrate targetare preferably ingested by the invertebrate as larger transcripts, thatis, larger than the 21 to 24 nucleotide fragments typically resultingfrom in planta processing. Thus, RNAi transcripts intended for ingestionare preferably designed to be resistant to in planta processing. Therecombinant invertebrate miRNAs of this invention are preferablyresistant to the plant-specific endogenous miRNA processing (incomparison to plant-derived miRNAs), but are preferably readilyrecognized in invertebrate cells where they are processed to the maturemiRNA.

SUMMARY OF THE INVENTION

In one aspect, this invention provides a non-natural plant havingresistance to an invertebrate pest that ingests RNA from the plant,wherein the plant includes a transgenic plant cell having in its genomea recombinant DNA construct that is transcribed in the transgenic plantcell to a recombinant miRNA precursor; and the recombinant miRNAprecursor includes a single strand of RNA that folds into the secondarystructure of an invertebrate miRNA precursor and that includes at leastone stem-loop that is processed to a mature miRNA; and the mature miRNAsuppresses expression of at least one target gene of the invertebratepest (or of a symbiont that is associated with the invertebrate pest),thereby conferring on the non-natural plant resistance to theinvertebrate pest.

Another aspect of this invention provides the recombinant DNA constructthat is transcribed in the non-natural transgenic plant cell to arecombinant miRNA precursor. In many embodiments, the recombinant DNAconstruct further includes one or more elements selected from: (a) apromoter functional in a plant cell; (b) a transgene transcription unit;(c) a gene suppression element; and (d) a transcriptionregulatory/transcript stabilizing element.

In a further aspect, this invention provides a non-natural transgenicplant cell having in its genome recombinant DNA that is transcribed inthe non-natural transgenic plant cell to a recombinant miRNA precursor,wherein the recombinant miRNA precursor includes a single strand of RNAthat folds into the secondary structure of an invertebrate miRNAprecursor and that includes at least one stem-loop that is processed toa mature miRNA, and wherein the mature miRNA suppresses expression of atleast one target gene of an invertebrate (or of a symbiont that isassociated with the invertebrate pest). Also provided are a non-naturaltransgenic plant containing the transgenic plant cell of this invention,a non-natural transgenic plant grown from the transgenic plant cell ofthis invention, and non-natural transgenic seed produced by thetransgenic plants, as well as commodity products produced from anon-natural transgenic plant cell, plant, or seed of this invention.

Other specific embodiments of the invention are disclosed in thefollowing detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a non-limiting example of multiple invertebrate miRNAprecursors, a Drosophila melanogaster “8-miR” sequence (SEQ ID NO. 1),as described in Example 1. The individual miRNA precursors indicated bybold underlined text.

FIG. 2 depicts the fold-back structure (that is, the secondary structureof the miRNA precursor including a stem-loop that is processed to themature miRNA) for each of the 8 miRNA precursors of the Drosophilamelanogaster “8-miR” sequence described in Example 1 and Table 1. Themature miRNA is indicated within the fold-back structure in boldcapitals. The stem region of the fold-back structure is generallyindicated by the vertical hatch marks indicating base pairing betweenthe first and second segments of the folded strand; the loop region isdepicted in each case to the right of the stem region. This illustratesnon-limiting embodiments of mismatches (see paragraph 0024), wherein atleast one nucleotide of the first segment is unpaired within thepartially double stranded RNA formed by hybridization of the first andsecond segments that are included in the stem region. One example of amismatch is the at least one extra or at least one missing nucleotide onthe second segment at the position corresponding to the nucleotide inquestion of the first segment in dme-mir-5 (SEQ ID NO. 15). Anotherexample of a mismatch is illustrated by the multiple non-base-pairednucleotides in dme-mir-309 (SEQ ID NO. 11).

FIG. 3 depicts native miRNA precursors and for each, the correspondingengineered miRNA precursor, as listed in Table 2 and described inExample 1. Secondary structure (i.e., fold-back structure) in theengineered miRNA precursor is preferably maintained to be similar tothat of the corresponding native miRNA precursor. Note that loop regionsmay include more than a single single-stranded segment (see thestructures of SEQ ID NO. 13 and SEQ ID NO. 38).

FIG. 4 depicts a Northern blot used to test in planta stability of anengineered miRNA precursor, as described in Example 1. “+” indicates RNAfrom plants transformed with pMON97878; “−” indicates negative controls.“8mirvATPase-16” (SEQ ID NO. 46) RNA was present in tobacco plants asboth full-length “8mirvATPase-16” transcript and as degraded RNA,demonstrating that “8mirvATPase-16” RNA is more stable in plants than isthe corresponding double-stranded RNA produced from an inverted repeat(i.e., sense adjacent to anti-sense of the same target gene), which wasfound to be entirely cleaved to small RNAs in planta (data not shown).

FIG. 5 depicts non-limiting examples of fold-back structures (i.e.,secondary structures of invertebrate miRNA precursors listed in Table 4,each including at least one stem-loop that is processed to a maturemiRNA, wherein the stem-loop includes a stem region and a loop region),as described in Example 2.

FIG. 6A depicts a native “SCN15” pre-miRNA sequence (SEQ ID NO. 57) ofwhich the complementary region of the foldback was changed to maintainthe original paired and unpaired bases, yielding the correspondingengineered pre-miRNA sequence “SCN15-MIRMSP1” (SEQ ID NO. 92), asdescribed in Example 4. FIG. 6B depicts a native “SCN25” pre-miRNAsequence (SEQ ID NO. 58) of which the complementary region of thefoldback was changed to maintain the original paired and unpaired bases,yielding the corresponding engineered pre-miRNA sequence “SCN25-MIRcgh1”(SEQ ID NO. 96), as described in Example 5.

FIG. 7 schematically depicts non-limiting recombinant DNA constructs asdescribed in Example 6. For use in Agrobacterium-mediated transformationof plant cells, at least one T-DNA border is generally included in eachconstruct (not shown). These constructs include a promoter element(“pro”), an intron flanked on one or on both sides by non-protein-codingDNA, an optional terminator element (“ter”), at least one first genesuppression element (“GSE” or “GSE1”) for suppressing at least one firsttarget gene, and can optionally include at least one second genesuppression element (“GSE2”) for suppressing at least one second targetgene, at least one gene expression element (“GEE”) for expressing atleast one gene of interest, or both. In embodiments containing a geneexpression element, the gene expression element can be located adjacentto (outside of) the intron. In one variation of this embodiment (notshown), the gene suppression element (embedded in an intron flanked onone or on both sides by non-protein-coding DNA) is located 3′ to theterminator. In other constructs of the invention (not shown), a genesuppression element (not intron-embedded) is located 3′ to theterminator.

FIG. 8 depicts various non-limiting examples of gene suppressionelements as described in Example 6. Where drawn as a single strand(FIGS. 6A through 6E), these are conventionally depicted in 5′ to 3′(left to right) transcriptional direction, where the arrows indicateanti-sense sequence (arrowhead pointing to the left), or sense sequence(arrowhead pointing to the right). Where drawn as double-stranded(anti-parallel) transcripts (FIGS. 6F and 6G), the 5′ and 3′transcriptional directionality is as shown. Solid lines, dashed lines,and dotted lines indicate sequences that target different target genes.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. Generally, the nomenclature usedand the manufacture or laboratory procedures described below are wellknown and commonly employed in the art. Conventional methods are usedfor these procedures, such as those provided in the art and variousgeneral references. Unless otherwise stated, nucleic acid sequences inthe text of this specification are given, when read from left to right,in the 5′ to 3′ direction. Nucleic acid sequences may be provided as DNAor as RNA, as specified; disclosure of one necessarily defines theother, as is known to one of ordinary skill in the art. Where a term isprovided in the singular, the inventors also contemplate aspects of theinvention described by the plural of that term. The nomenclature usedand the laboratory procedures described below are those well known andcommonly employed in the art. Where there are discrepancies in terms anddefinitions used in references that are incorporated by reference, theterms used in this application shall have the definitions given. Othertechnical terms used have their ordinary meaning in the art that theyare used, as exemplified by a variety of technical dictionaries. Theinventors do not intend to be limited to a mechanism or mode of action.Reference thereto is provided for illustrative purposes only.

Plant Having Resistance to an Invertebrate Pest

In one aspect, this invention provides a non-natural plant havingresistance to an invertebrate pest that ingests RNA from the plant,wherein the plant includes a transgenic plant cell having in its genomea recombinant DNA construct that is transcribed in the transgenic plantcell to a recombinant miRNA precursor; and the recombinant miRNAprecursor includes a single strand of RNA that folds into the secondarystructure of an invertebrate miRNA precursor and that includes at leastone stem-loop that is processed to a mature miRNA; and the mature miRNAsuppresses expression of at least one target gene of the invertebratepest (or of a symbiont that is associated with the invertebrate pest),thereby conferring on the non-natural plant resistance to theinvertebrate pest.

In another aspect, the recombinant DNA construct that is transcribed inthe transgenic plant cell to a recombinant miRNA precursor is alsospecifically disclosed and claimed herein. The recombinant DNA constructis made by techniques known in the art, such as those described underthe heading “Making and Using Recombinant DNA Constructs” andillustrated in the working Examples. The recombinant DNA construct isparticularly useful for making transgenic plant cells, transgenicplants, and transgenic seeds as discussed below under “Making and UsingTransgenic Plant Cells and Transgenic Plants”. The recombinant miRNAprecursor is generally transcribed as a single strand of RNA. Thissingle strand of RNA includes at least one stem-loop that can beregarded as equivalent to a naturally occurring pre-miRNA, in that it isprocessed to a mature miRNA. The stem-loop is formed when the singlestrand folds back on itself and sufficient base pairing occurs tostabilize the resulting folded structure. The stem-loop includes a stemregion and a loop region, all within the same single strand of RNA. Thestem region includes a first segment and a second segment, which arejoined through the loop region. Note that the loop region can includestructures more complex than a simple single-stranded loop, anon-limiting example of which is illustrated by mir286 (SEQ ID NO. 13)and its engineered counterpart (SEQ ID NO. 38) in FIG. 3. The firstsegment includes at least 19 contiguous nucleotides for silencing amessenger RNA encoding the target gene. The second segment contains atleast 19 contiguous nucleotides. The first segment and second segmentare generally of similar length (in terms of number of contiguousnucleotides making up each segment) but are not necessarily of identicallength. The loop region is located on the single strand between thefirst and second segments. The first and second segments hybridize toform partially double stranded RNA, wherein at least one nucleotide ofthe first segment is unpaired; that is, within the partially doublestranded RNA, there is at least one nucleotide of the first segment thatis mismatched to the corresponding position on the second segment. Themismatch results in a bulge or loop or kink in the otherwisesubstantially double-stranded stem region. The mismatch can be due to,for example, at least one nucleotide of the second segment that does notbase-pair to the nucleotide in question of the first segment, or atleast one extra or at least one missing nucleotide on the second segmentat the position corresponding to the nucleotide in question of the firstsegment. FIG. 2 illustrates non-limiting examples of mismatches.

The first segment of the stem region includes at least 19 contiguousnucleotides for silencing a messenger RNA encoding the target gene (the“target mRNA”); the mature miRNA that is processed from the stem-loopincludes these at least 19 contiguous nucleotides. These at least 19contiguous nucleotides have at least about 70% complementarity to asegment of equivalent length (that is, at least about 70%complementarity to a segment having about the same number of contiguousnucleotides) in the target mRNA. For example, where the first segment ofthe stem region consists of exactly 19 contiguous nucleotides forsilencing the target mRNA, these 19 contiguous nucleotides can include13 nucleotides (13/19=68% complementarity), 14 nucleotides (14/19=74%complementarity), 15 nucleotides (15/19=79% complementarity), 16nucleotides (16/19=84% complementarity), 17 nucleotides (17/19=89%complementarity), 18 nucleotides (18/19=95% complementarity), or even 19nucleotides (19/19=100% complementarity) that are complementary to a19-nucleotide segment in the target mRNA.

In preferred embodiments, the at least 19 contiguous nucleotides have atleast about 75%, or at least about 80%, or at least about 85%, or atleast about 90% complementarity to a segment of equivalent length in thetarget mRNA. In one particularly preferred embodiment, the at least 19contiguous nucleotides have at least about 95% complementarity to asegment of equivalent length in the target mRNA. In another particularlypreferred embodiment, the at least 19 contiguous nucleotides have 100%complementarity to a segment of equivalent length in the target mRNA.

The degree of complementarity between the stem region's first segment'sat least 19 contiguous nucleotides to a segment of equivalent length inthe target mRNA is readily selected by one of skill in the art. It hasbeen reported that base pairing between nucleotides located towards the5′ end of the mature miRNA to the target mRNA is comparatively importantin the ability of a mature miRNA to silence expression of the targetmRNA (see, for example, Doensch and Sharp (2004) Genes Dev.,18:504-511), and it is expected that high complementarity between the 5′end of the mature miRNA to the target can allow a relatively higherdegree of mismatch between nucleotides closer to the 3′ end of themature miRNA and the target (and vice versa). Thus, in a preferredembodiment, the nucleotide sequence of the stem region's first segment'sat least 19 contiguous nucleotides is selected so that the mature miRNAprocessed from the stem-loop is perfectly complementary to the targetmRNA at the 5′-most 8 nucleotides of the mature miRNA. In anotherpreferred embodiment, the nucleotide sequence of the stem region's firstsegment's at least 19 contiguous nucleotides is selected so that themature miRNA processed from the stem-loop is perfectly complementary tothe target mRNA at nucleotide positions 2, 3, 4, 5, 6, and 7 (from the5′ end) of the mature miRNA. In another preferred embodiment, thenucleotide sequence of the stem region's first segment's at least 19contiguous nucleotides is designed so that the mature miRNA processedfrom the stem-loop structure has few or no G:U wobble base pairs.

The loop region of the stem-loop typically includes between about 4 toabout 40 nucleotides. In some preferred embodiments, the loop regionincludes consecutive nucleotides of a native loop sequence of theinvertebrate miRNA precursor. In some embodiments, the loop region isidentical to a native loop sequence of the invertebrate miRNA precursor.

In some embodiments, the stem-loop is processed to a mature miRNA,typically of 21, 22, 23, 24, 25, or 26 nucleotides in length, in thetransgenic plant cell. In other embodiments, the stem-loop preferablyremains relatively intact (that is, substantially uncleaved to smallerpolynucleotides) in the transgenic plant cell, but is processed to amature miRNA (typically of 21, 22, 23, 24, 25, or 26 nucleotides inlength) in the gut or in or on a cell of an invertebrate that ingestsRNA from the plant that includes the transgenic plant cell.

In one embodiment, the single strand of RNA includes a single stem-loopthat is processed to a mature miRNA. In other embodiments, the singlestrand of RNA includes multiple stem-loops that are processed to maturemiRNAs. Where multiple stem-loops are present, they can consist ofmultiples of the same stem-loop, or multiple different stem-loops. Inone preferred embodiment, the single strand of RNA includes multiplestem-loops that correspond to a group of invertebrate miRNAs that arenatively transcribed in a single polycistronic transcription unit. Onenon-limiting example is a single strand of RNA including multiplestem-loops that correspond to a polycistronic group of 8 miRNAs found onChromosome 2R in Drosophila melanogaster (SEQ ID NO. 1, see Example 1).

The target gene of the invertebrate pest (or of a symbiont that isassociated with the invertebrate pest) that is suppressed by the maturemiRNA can be a single target gene, or can be multiple target genes(e.g., multiple alleles of a given target gene, or multiple unrelatedtarget genes). Target genes of interest are described in detail in thesection “Target Genes and Pest Invertebrates”.

In some embodiments, the at least one target gene is an endogenous ornative target of an invertebrate miRNA natively expressed from theinvertebrate miRNA precursor. In these embodiments, the mature miRNAprocessed from the stem-loop is identical (or nearly identical) to themature miRNA natively processed from the naturally occurringinvertebrate miRNA precursor. Generally, therefore, the recombinantmiRNA precursor is substantially similar to the invertebrate miRNAprecursor, although the recombinant miRNA precursor is designed toexpress the mature miRNA generally under non-native conditions. In anon-limiting example, the recombinant DNA encodes a native invertebratemiRNA precursor, expressed under the control of a promoter that differsfrom the native promoter of the invertebrate miRNA precursor.

In other embodiments, the at least one target gene is other than anendogenous target of an invertebrate miRNA natively expressed from theinvertebrate miRNA precursor. In these embodiments, the mature miRNAprocessed from the stem-loop is an “engineered miRNA”, that is, a maturemiRNA having an artificial sequence designed to suppress a target geneof choice. Factors considered in the design of such an engineered miRNAsequence are described in detail in the section “Target Genes and PestInvertebrates”, in the working examples, and elsewhere in thisdisclosure.

The invertebrate pest is at least one or more invertebrate selected fromthe group consisting of insects, arachnids (e.g., mites), nematodes,molluscs (e.g., slugs and snails), and annelids, and can include aninvertebrate associated with an invertebrate pest in a symbioticrelationship (e.g., the mutualistic relationship between some ant andaphid species). The term “symbiotic” relationship as used hereinencompasses both facultative (non-obligate) and obligate symbioseswherein at least one of the two or more associated species benefits, andfurther includes mutualistic, commensal, and parasitic relationships.Symbionts also include non-invertebrate symbionts, such as prokaryotesand eukaryotic protists. An invertebrate pest can be controlledindirectly by targetting a symbiont that is associated, internally orexternally, with the invertebrate pest. For example, prokaryoticsymbionts are known to occur in the gut or other tissues of manyinvertebrates, including invertebrate pests of interest. Non-limitingexamples of a targetted symbiont associated with an invertebrate pestinclude the aphid endosymbiotic bacteria Buchnera; Wolbachia bacteriathat infect many insects; Baumannia cicadellinicola and Sulcia muelleri,the co-symbiotic bacteria of the glassy-winged sharpshooter (Homalodiscacoagulata), which transmits the Pierce's disease pathogen Xylellafastidiosa; and eukaryotic protist (flagellate) endosymbionts intermites. Also see, for example, Wu et al. (2006) PLoS Biol., 4(6):c188doi:10.1371/journal.pbio.0040188; Moran and Telang (1998) BioScience,48:295-304; and Moran and Baumann (2000) Curr. Opin. Microbiol.,3:270-275. In an alternative approach, expression of an endogenoustarget gene of the invertebrate pest can be modified in such a way as tocontrol a symbiont of the invertebrate, in turn affecting the hostinvertebrate. For example, it was reported that RNAi-mediated genesuppression using constructs (head-to-tail inverted repeats of thetarget gene including an intronic spacer) targetting the Drosophilahomeobox gene Caudal, which represses nuclear factor kappa B-dependentantimicrobial peptide genes, led to overexpression of antimicrobialpeptides, thus altering the commensal bacterial population in theDrosophila gut and eventually leading to gut cell apoptosis and hostmortality; see Ryu et al (2008) Science, 319:777-782.

Pests of interest are described in detail in the section “Target Genesand Pest Invertebrates”. Of particular interest are sapsucking insects,such as aphids, Lygus, leafhoppers, whiteflies, thrips, scale insectsand mealybugs, as well as insects that ingest plant tissues or cells,such as lepidopteran larvae. Non-limiting embodiments includeembodiments where (a) the plant is maize and the invertebrate pest is aDiabrotica species; (b) the plant is soybean and the invertebrate pestis a soybean cyst nematode (Heterodera glycines); (c) the plant is agrape and the invertebrate pest is a grape phylloxera or a glassy-wingedsharpshooter (Homalodisca coagulata); and (d) the plant is an apple treeand the invertebrate pest is a woolly apple aphid.

In many embodiments, the recombinant DNA construct further includes oneor more elements selected from: (a) a promoter functional in a plantcell; (b) a transgene transcription unit; (c) a gene suppressionelement; and (d) a transcription regulatory/transcript stabilizingelement. Promoters useful in this invention have promoter activity in aplant cell. Suitable promoters include those described in detail underthe heading “Promoters”. Non-limiting examples of promoters includeconstitutive promoters, promoters with expression patterns in tissueslikely to be contacted by the pest invertebrate (e.g., phloem-specificpromoters, vascular-specific promoters, or root-specific promoters), andinducible promoters such as promoters that are induced by stress(abiotic stress or biotic stress such as stress from an infestation bythe pest invertebrate).

A transgene transcription unit includes DNA sequence encoding a gene ofinterest. A gene of interest can include any coding or non-codingsequence from any species (including, but not limited to, non-eukaryotessuch as bacteria, and viruses; fungi; protists, including protozoans;plants, including monocots and dicots, such as crop plants, ornamentalplants, and non-domesticated or wild plants; invertebrates such asarthropods, annelids, nematodes, and molluscs; and vertebrates such asamphibians, fish, birds, and mammals. Non-limiting examples of anon-coding sequence to be expressed by a transgene transcription unitinclude, but not limited to, 5′ untranslated regions, promoters,enhancers, or other non-coding transcriptional regions, 3′ untranslatedregions, terminators, intron, microRNAs, microRNA precursor DNAsequences, small interfering RNAs, RNA components of ribosomes orribozymes, small nucleolar RNAs, RNA aptamers capable of binding to aligand, and other non-coding RNAs. Non-limiting examples of a gene ofinterest further include, but are not limited to, translatable (coding)sequence, such as genes encoding transcription factors and genesencoding enzymes involved in the biosynthesis or catabolism of moleculesof interest (such as amino acids, fatty acids and other lipids, sugarsand other carbohydrates, biological polymers, and secondary metabolitesincluding alkaloids, terpenoids, polyketides, non-ribosomal peptides,and secondary metabolites of mixed biosynthetic origin). A gene ofinterest can be a gene native to the cell (e.g., a plant cell) in whichthe recombinant DNA construct of the invention is to be transcribed, orcan be a non-native gene. A gene of interest can be a marker gene, forexample, a selectable marker gene encoding antibiotic, antifungal, orherbicide resistance, or a marker gene encoding an easily detectabletrait (e.g., in a plant cell, phytoene synthase or other genes impartinga particular pigment to the plant), or a gene encoding a detectablemolecule, such as a fluorescent protein, luciferase, or a uniquepolypeptide or nucleic acid “tag” detectable by protein or nucleic aciddetection methods, respectively). Selectable markers are genes ofinterest of particular utility in identifying successful processing ofconstructs of the invention. Genes of interest include those genes alsodescribed above as target genes, under the heading “Target Genes”. Thetransgene transcription unit can further include 5′ or 3′ sequence orboth as required for transcription of the transgene.

Gene suppression elements include any DNA sequence (or RNA sequenceencoded therein) designed to specifically suppress a gene or genes ofinterest. In the context of “gene suppression elements”, “target gene”generally refers to a gene other than the target gene of theinvertebrate pest silenced by the mature miRNA of this invention; thiscan be a gene endogenous to the transgenic plant cell or a geneexogenous to the plant. In the context of “gene suppression elements”,non-limiting examples of suitable target genes also include amino acidcatabolic genes (such as, but not limited to, the maize LKRISDH geneencoding lysine-ketoglutarate reductase (LKR) and saccharopinedehydrogenase (SDH), and its homologues), maize zein genes, genesinvolved in fatty acid synthesis (e.g., plant microsomal fatty aciddesaturases and plant acyl-ACP thioesterases, such as, but not limitedto, those disclosed in U.S. Pat. Nos. 6,426,448, 6,372,965, and6,872,872), genes involved in multi-step biosynthesis pathways, where itmay be of interest to regulate the level of one or more intermediates,such as genes encoding enzymes for polyhydroxyalkanoate biosynthesis(see, for example, U.S. Pat. No. 5,750,848); and genes encodingcell-cycle control proteins, such as proteins with cyclin-dependentkinase (CDK) inhibitor-like activity (see, for example, genes disclosedin International Patent Application Publication Number WO 05007829A2).Target genes can include genes encoding undesirable proteins (e.g.,allergens or toxins) or the enzymes for the biosynthesis of undesirablecompounds (e.g., undesirable flavor or odor components). Thus, oneembodiment of the invention is a transgenic plant or tissue of such aplant that is further improved by the suppression of allergenic proteinsor toxins, e.g., a peanut, soybean, or wheat kernel with decreasedallergenicity. Target genes can include genes involved in fruitripening, such as polygalacturonase. Target genes can include geneswhere expression is preferably limited to a particular cell or tissue ordevelopmental stage, or where expression is preferably transient, thatis to say, where constitutive or general suppression, or suppressionthat spreads through many tissues, is not necessarily desired. Thus,other examples of suitable target genes include genes encoding proteinsthat, when expressed in transgenic plants, make the transgenic plantsresistant to pests or pathogens (see, for example, genes for cholesteroloxidase as disclosed in U.S. Pat. No. 5,763,245); genes where expressionis pest- or pathogen-induced; and genes which can induce or restorefertility (see, for example, the barstar/barnase genes described in U.S.Pat. No. 6,759,575); all the patents cited in this paragraph areincorporated by reference in their entirety herein.

Suitable gene suppression elements are described in detail in U. S.Patent Application Publication 2006/0200878, which is incorporatedherein by reference, and include one or more of:

-   -   (a) DNA that includes at least one anti-sense DNA segment that        is anti-sense to at least one segment of the target gene;    -   (b) DNA that includes multiple copies of at least one anti-sense        DNA segment that is anti-sense to at least one segment of the        target gene;    -   (c) DNA that includes at least one sense DNA segment that is at        least one segment of the target gene;    -   (d) DNA that includes multiple copies of at least one sense DNA        segment that is at least one segment of the target gene;    -   (e) DNA that transcribes to RNA for suppressing the target gene        by forming double-stranded RNA and includes at least one        anti-sense DNA segment that is anti-sense to at least one        segment of the target gene and at least one sense DNA segment        that is at least one segment of the target gene;    -   (f) DNA that transcribes to RNA for suppressing the target gene        by forming a single double-stranded RNA and includes multiple        serial anti-sense DNA segments that are anti-sense to at least        one segment of the target gene and multiple serial sense DNA        segments that are at least one segment of the target gene;    -   (g) DNA that transcribes to RNA for suppressing the target gene        by forming multiple double strands of RNA and includes multiple        anti-sense DNA segments that are anti-sense to at least one        segment of the target gene and multiple sense DNA segments that        are at least one segment of the target gene, and wherein the        multiple anti-sense DNA segments and the multiple sense DNA        segments are arranged in a series of inverted repeats;    -   (h) DNA that includes nucleotides derived from a plant miRNA;    -   (i) DNA that includes nucleotides of a siRNA;    -   (j) DNA that transcribes to an RNA aptamer capable of binding to        a ligand; and    -   (k) DNA that transcribes to an RNA aptamer capable of binding to        a ligand, and DNA that transcribes to regulatory RNA capable of        regulating expression of the target gene, wherein the regulation        is dependent on the conformation of the regulatory RNA, and the        conformation of the regulatory RNA is allosterically affected by        the binding state of the RNA aptamer.

DNA elements for suppressing expression are described further in Example6 and depicted in FIGS. 7 and 8.

Transcription regulatory elements include elements that regulate theexpression level of the recombinant DNA construct of this invention(relative to its expression in the absence of such regulatory elements).Non-limiting examples of suitable transcription regulatory elementsinclude riboswitches (cis- or trans-acting) and miRNA recognition sites,as described in detail in U. S. Patent Application Publication2006/0200878, incorporated herein by reference. Other examples oftranscription regulatory elements include transcript stabilizingelements such as an RNA that assumes a secondary structure orthree-dimensional configuration (e.g., a loop, stem-loop, pseudoknot)that confers on the transcript increased stability or increasedhalf-life in vivo; an RNA aptamer that confers on the transcriptincreased cell or tissue specificity; and transcript destabilizingelements such as the SAUR destabilizing sequences described in detail inU. S. Patent Application Publication 2007/0011761, incorporated hereinby reference.

In some embodiments of this invention, the non-natural plant is anon-natural transgenic plant, such as one provided by techniquesdescribed below under the heading “Making and Using Transgenic PlantCells and Transgenic Plants”. In such embodiments, all cells (with thepossible exception of haploid cells) and tissues of the non-naturalplant contain the recombinant DNA construct of this invention.

In other embodiments, the non-natural plant is not completelytransgenic, but includes natural non-transgenic tissue (for example,non-natural transgenic tissue grafted onto natural non-transgenictissue). In a non-limiting embodiment, the non-natural plant includes anatural non-transgenic scion and a non-natural transgenic rootstockincluding the transgenic plant cell, wherein the non-transgenic scionand transgenic rootstock are grafted together. Such embodiments areparticularly useful where the plant is one that is commonly vegetativelygrown as a scion grafted onto a rootstock (wherein scion and rootstockcan be of the same species or variety or of different species orvariety); non-limiting examples include grapes (e.g., wine grapes andtable grapes), apples, pears, quince, avocados, citrus, stone fruits(e.g., peaches, plums, nectarines, apricots, cherries), kiwifruit,roses, and other plants of agricultural or ornamental importance.Specifically claimed embodiments include embodiments where (a) thenon-natural plant includes a natural non-transgenic grape scion and anon-natural transgenic grape rootstock and the invertebrate pest is agrape phylloxera; and (b) the non-natural plant includes a naturalnon-transgenic fruit tree (e.g., pear) scion and a non-naturaltransgenic fruit tree (e.g., quince) rootstock.

Target Genes and Pest Invertebrates

In one aspect, this invention provides a recombinant DNA construct thatis transcribed in the transgenic plant cell to a recombinant miRNAprecursor; wherein the recombinant miRNA precursor includes a singlestrand of RNA that folds into the secondary structure of an invertebratemiRNA precursor and that includes at least one stem-loop that isprocessed to a mature miRNA; and the mature miRNA suppresses expressionof at least one target gene of the invertebrate pest, thereby conferringon the plant resistance to the invertebrate pest. The target gene of theinvertebrate pest can be any target gene or genes of the invertebratepest. The target gene can be a target gene of a symbiont associated withthe invertebrate pest; suppression of such a symbiont gene confers onthe plant resistance to the invertebrate pest. The target gene caninclude a single gene or part of a single gene that is targetted forsuppression, or can include, for example, multiple consecutive segmentsof a target gene, multiple non-consecutive segments of a target gene,multiple alleles of a target gene, or multiple target genes from one ormore species.

A target gene includes a sequence endogenous to any invertebrate pestspecies (or of a symbiont associated with the invertebrate pest). Ofparticular interest are arthropods (insects and arachnids), nematodes,molluscs (such as slugs or snails), annelids, and obligate symbionts ofinvertebrate pests. The target gene can be translatable (coding)sequence, or can be non-coding sequence (such as non-coding regulatorysequence), or both. Non-limiting examples of a target gene includenon-translatable (non-coding) sequence, such as, but not limited to, 5′untranslated regions, promoters, enhancers, or other non-codingtranscriptional regions, 3′ untranslated regions, terminators, andintrons. Target genes include genes encoding microRNAs (that is, theprimary transcript encoding an endogenous microRNA, or the RNAintermediates processed from this primary transcript), small interferingRNAs, RNA components of ribosomes or ribozymes, small nucleolar RNAs,and other non-coding RNAs (see, for example, non-coding RNA sequencesprovided publicly at rfam.wustl.edu; Erdmann et al. (2001) Nucleic AcidsRes., 29:189-193; Gottesman (2005) Trends Genet., 21:399-404;Griffiths-Jones et al. (2005) Nucleic Acids Res., 33:121-124). Targetgenes can also include translatable (coding) sequence for genes encodingtranscription factors and genes encoding enzymes involved in thebiosynthesis or catabolism of molecules of interest (such as, but notlimited to, amino acids, fatty acids and other lipids, sugars and othercarbohydrates, biological polymers, and secondary metabolites includingalkaloids, terpenoids, polyketides, non-ribosomal peptides, andsecondary metabolites of mixed biosynthetic origin).

In many preferred embodiments, the target gene is an essential gene ofthe invertebrate pest (or of a symbiont associated with the invertebratepest). Essential genes include genes that are required for developmentof the invertebrate pest to a fertile reproductive adult. Essentialgenes include genes that, when silenced or suppressed, result in thedeath of the invertebrate pest (as an adult or at any developmentalstage, including gametes) or in the invertebrate pest's inability tosuccessfully reproduce (e.g., sterility in a male or female parent orlethality to the zygote, embryo, or larva). A description of nematodeessential genes is found, e.g., in Kemphues, K. “Essential Genes” (Dec.24, 2005), WormBook, ed. The C. elegans Research Community, WormBook,doi/10.1895/wormbook.1.57.1, available on line at www.wormbook.org.Soybean cyst nematode essential genes are disclosed in U.S. patentapplication Ser. No. 11/360,355, filed 23 Feb. 2006, incorporated byreference herein. Non-limiting examples of invertebrate essential genesinclude major sperm protein, alpha tubulin, beta tubulin, vacuolarATPase, glyceraldehyde-3-phosphate dehydrogenase, RNA polymerase II,chitin synthase, cytochromes, miRNAs, miRNA precursor molecules, miRNApromoters, as well as other genes such as those disclosed in U. S.Patent Application Publication 2006/0021087 A1, PCT Patent ApplicationPCT/US05/11816, and in Table II of U. S. Patent Application Publication2004/0098761 A1, which are incorporated by reference herein. Adescription of insect genes is publicly available at the Drosophilagenome database (available on line at flybase.bio.indiana.edu/). Themajority of predicted Drosophila genes have been analyzed for functionby a cell culture-based RNA interference screen, resulting in 438essential genes being identified; see Boutros et al. (2004) Science,303:832-835, and supporting material available on line atwww.sciencemag.org/cgi/content/full/303/5659/832/DC1. Other examples ofessential insect genes include a gut cell protein, a membrane protein,an ecdysone receptor, ATPases such as gamma-ATPase, an amino acidtransporter, a transcription factor, a peptidylglycine alpha-amidatingmonooxygenase; a cysteine protease, an aminopeptidase, a dipeptidase, asucrase/transglucosidase, a translation elongation factor, an eukaryotictranslation initiation factor 1A, a splicing factor, an apoptosisinhibitor; a tubulin protein, an actin protein, an alpha-actininprotein, a histone, a histone deacetylase, a cell cycle regulatoryprotein, a cellular respiratory protein; a receptor for aninsect-specific hormonal signal, a juvenile hormone receptor, an insectpeptidic hormone receptor; a protein regulating ion balance in a cell, aproton-pump, a Na/K pump, an intestinal protease; an enzyme involved insucrose metabolism, a digestive enzyme, a trypsin-like protease and acathepsin B-like protease. Essential genes include those that influenceother genes, where the overall effect is the death of the invertebratepest or loss of the invertebrate pest's inability to successfullyreproduce. In an non-limiting example, suppression of the Drosophilahomeobox gene Caudal leads eventually to host mortality caused by anindirect effect (i.e., the disequilibrium of the insect's commensal gutbacterial population) (Ryu et al (2008) Science, 319:777-782) and thusCaudal as well as the antimicrobial peptide genes directly controlled byCaudal are both considered essential genes.

Plant pest invertebrates include, but are not limited to, nematodes,molluscs (slugs and snails), and insects and arachnids. See also G. N.Agrios, “Plant Pathology” (Fourth Edition), Academic Press, San Diego,1997, 635 pp., for descriptions of nematodes and flagellate protozoans,all of which are invertebrate pests of interest. See also thecontinually updated compilation of plant pests and the diseases causedby such on the American Phytopathological Society's “Common Names ofPlant Diseases”, compiled by the Committee on Standardization of CommonNames for Plant Diseases of The American Phytopathological Society,1978-2005, available online at www.apsnet.org/online/common/top.asp.

Non-limiting examples of invertebrate pests include cyst nematodesHeterodera spp. especially soybean cyst nematode Heterodera glycines,root knot nematodes Meloidogyne spp., lance nematodes Hoplolaimus spp.,stunt nematodes Tylenchorhynchus spp., spiral nematodes Helicotylenchusspp., lesion nematodes Pratylenchus spp., ring nematodes Criconema spp.,foliar nematodes Aphelenchus spp. or Aphelenchoides spp., cornrootworms, Lygus spp., aphids and similar sap-sucking insects such asphylloxera (Daktulosphaira vitifoliae), corn borers, cutworms,armyworms, leafhoppers, Japanese beetles, grasshoppers, and other pestcoleopterans, dipterans, and lepidopterans. Specific examples ofinvertebrate pests include pests capable of infesting the root systemsof crop plants, e.g., northern corn rootworm (Diabrotica barberi),southern corn rootworm (Diabrotica undecimpunctata), Western cornrootworm (Diabrotica virgifera), corn root aphid (Anuraphismaidiradicis), black cutworm (Agrotis ipsilon), glassy cutworm (Crymodesdevastator), dingy cutworm (Feltia ducens), claybacked cutworm (Agrotisgladiaria), wireworm (Melanotus spp., Aeolus mellillus), wheat wireworm(Aeolus mancus), sand wireworm (Horistonotus uhlerii), maize billbug(Sphenophorus maidis), timothy billbug (Sphenophorus zeae), bluegrassbillbug (Sphenophorus parvulus), southern corn billbug (Sphenophoruscallosus), white grubs (Phyllophaga spp.), seedcorn maggot (Deliaplatura), grape colaspis (Colaspis brunnea), seedcorn beetle(Stenolophus lecontei), and slender seedcorn beetle (Cliviniaimpressifrons), as well as the parasitic nematodes listed in Table 6 ofU.S. Pat. No. 6,194,636, which is incorporated in its entirety byreference herein.

Invertebrate pests of particular interest, especially in but not limitedto southern hemisphere regions (including South and Central America)include aphids, corn rootworms, spodoptera, noctuideae, potato beetle,Lygus spp., any hemipteran, homopteran, or heteropteran, anylepidopteran, any coleopteran, nematodes, cutworms, earworms, armyworms,borers, leaf rollers, and others. Arthropod pests specificallyencompassed by this invention include various cutworm species includingcutworm (Agrotis repleta), black cutworm (Agrotis ipsilon), cutworm(Anicla ignicans), granulate cutworm (Feltia subterranea), “gusanoaspero” (Agrotis malefida); Mediterranean flour moth (Anagastakuehniella), square-necked grain beetle (Cathartus quadricollis), fleabeetle (Chaetocnema spp), rice moth (Corcyra cephalonica), corn rootwormor “vaquita de San Antonio” (Diabotica speciosa), sugarcane borer(Diatraea saccharalis), lesser cornstalk borer (Elasmopalpuslignosellus), brown stink bug (Euschistus spp.), corn earworm(Helicoverpa zea), flat grain beetle (Laemophloeus minutus), grasslooper moth (Mocis latipes), sawtoothed grain beetle (Oryzaephilussurinamensis), meal moth (Pyralis farinalis), Indian meal moth (Plodiainterpunctella), corn leaf aphid (Rhopalosiphum maidis), brown burrowingbug or “chinche subterranea” (Scaptocoris castanea), greenbug(Schizaphis graminum), grain weevil (Sitophilus zeamais), Angoumoisgrain moth (Sitotroga cerealella), fall armyworm (Spodopterafrugiperda), cadelle beetle (Tenebroides mauritanicus), two-spottedspider mite (Tetranychus urticae), red flour beetle (Triboleumcastaneum), cotton leafworm (Alabama argillacea), boll weevil(Anthonomus grandis), cotton aphid (Aphis gossypii), sweet potatowhitefly (Bemisia tabaci), various thrips species (Frankliniella spp.),cotton earworm (Helicoverpa zea), “oruga bolillera” (e.g., Helicoverpageletopoeon), tobacco budworm (Heliothis virescens), stinkbug (Nezaraviridula), pink bollworm (Pectinophora gossypiella), beet armyworm(Spodoptera exigua), spider mites (Tetranychus spp.), onion thrips(Thrips tabaci), greenhouse whitefly (Trialeurodes vaporarium),velvetbean caterpillar (Anticarsia gemmatalis), spotted maize beetle or“astilo moteado” (Astylus atromaculatus), “oruga de la alfalfa” (Coliaslesbia), “chinche marrón” or “chinche de los cuernos” (Dichelopsfurcatus), “alquiche chico” (Edessa miditabunda), blister beetles(Epicauta spp.), “barrenador del brote” (Epinotia aporema), “oruga verdedel yuyo colorado” (Loxostege bifidalis), rootknot nematodes(Meloidogyne spp.), “oruga cuarteadora” (Mocis repanda), southern greenstink bug (Nezara viridula), “chinche de la alfalfa” (Piezodorusguildinii), green cloverworm (Plathypena scabra), soybean looper(Pseudoplusia includens), looper moth “isoca medidora del girasol”(Rachiplusia nu), yellow woolybear (Spilosoma virginica), yellowstripedarmyworm (Spodoptera ornithogalli), various root weevils (familyCurculionidae), various wireworms (family Elateridae), and various whitegrubs (family Scarabaeidae). Nematode pests specifically encompassed bythis invention include nematode pests of maize (Belonolaimus spp.,Trichodorus spp., Longidorus spp., Dolichodorus spp., Anguina spp.,Pratylenchus spp., Meloidogyne spp., Heterodera spp.), soybean(Heterodera glycines, Meloidogyne spp., Belonolaimus spp.), bananas(Radopholus similis, Meloidogyne spp., Helicotylenchus spp.), sugarcane(Heterodera sacchari, Pratylenchus spp., Meloidogyne spp.), oranges(Tylenchulus spp., Radopholus spp., Belonolaimus spp., Pratylenchusspp., Xiphinema spp.), coffee (Meloidogyne spp., Pratylenchus spp.),coconut palm (Bursaphelenchus spp.), tomatoes (Meloidogyne spp.,Belonolaimus spp., Nacobbus spp.), grapes (Meloidogyne spp., Xiphinemaspp., Tylenchulus spp., Criconemella spp.), lemon and lime (Tylenchulusspp., Radopholus spp., Belonolaimus spp., Pratylenchus spp., Xiphinemaspp.), cacao (Meloidogyne spp., Rotylenchulus reniformis), pineapple(Meloidogyne spp., Pratylenchus spp., Rotylenchulus reniformis), papaya(Meloidogyne spp., Rotylenchulus reniformis), grapefruit (Tylenchulusspp., Radopholus spp. Belonolaimus spp., Pratylenchus spp., Xiphinemaspp.), and broad beans (Meloidogyne spp.).

The recombinant DNA construct can be designed to be more specificallysuppress the target gene, for example, by designing the recombinant DNAconstruct to encode a recombinant miRNA precursor that is processed to amature miRNA that includes regions substantially non-complementary to anon-target gene sequence. Non-target genes can include any gene notintended to be silenced or suppressed, either in a plant containing therecombinant DNA construct or in organisms that may come into contactwith the recombinant DNA construct. A non-target gene sequence caninclude any sequence from any species (including, but not limited to,non-eukaryotes such as bacteria, and viruses; fungi; plants, includingmonocots and dicots, such as crop plants, ornamental plants, andnon-domesticated or wild plants; invertebrates such as arthropods,annelids, nematodes, and molluscs; and vertebrates such as amphibians,fish, birds, domestic or wild mammals, and even humans).

In one embodiment, the target gene is a gene endogenous to a specificinvertebrate pest species of interest, and the non-target gene is a geneor genes of one or more non-target species (such as a gene or genes of aplant species or a gene of a virus, fungus, bacterium, a non-targetinvertebrate, or vertebrate, even a human). One non-limiting example iswhere the recombinant DNA construct is designed to be processed to amature miRNA for suppressing a target gene that is a gene endogenous toa single species (e.g., Western corn rootworm, Diabrotica virgiferavirgifera LeConte) but not suppressing a non-target gene such as genesfrom related, even closely related, species (e.g., Northern cornrootworm, Diabrotica barberi Smith and Lawrence, or Southern cornrootworm, Diabrotica undecimpunctata).

In other embodiments (e.g., where it is desirable to suppress a targetgene across multiple species), it may be desirable to design therecombinant DNA construct to be processed to a mature miRNA forsuppressing a target gene sequence common to the multiple species inwhich the target gene is to be silenced. Thus, the miRNA processed fromthe recombinant DNA construct can be designed to be specific for onetaxon (for example, specific to a genus, family, or even a larger taxonsuch as a phylum, e.g., arthropoda) but not for other taxa (e.g., plantsor vertebrates or mammals). In one non-limiting example of thisembodiment, the recombinant DNA construct can be designed to beprocessed to a mature miRNA for suppressing a target gene sequencecommon to aphids (Aphidoidea) but not target any gene sequence fromother insects or invertebrates.

In another non-limiting example of this embodiment, a recombinant DNAconstruct for gene silencing in corn rootworm is designed to beprocessed to a mature miRNA for suppressing a target gene sequencecommon to all members of the genus Diabrotica. In a further example ofthis embodiment, such a Diabrotica-targetted recombinant DNA constructcan be selected so as to not target any sequence from beneficialcoleopterans (for example, predatory coccinellid beetles, commonly knownas ladybugs or ladybirds) or other beneficial insect species.

The required degree of specificity of a recombinant DNA construct ofthis invention for silencing a target gene depends on various factors.Factors can include the size and nucleic acid sequence of the maturemicroRNA encoded by the recombinant DNA construct, and the relativeimportance of decreasing such a mature miRNA's potential to suppressnon-target genes. In a non-limiting example, where such a mature miRNAis expected to be 22 base pairs in size, one particularly preferredembodiment includes DNA encoding a mature miRNA for silencing a targetgene wherein the mature miRNA includes sequence that is substantiallynon-identical to a non-target gene sequence, such as fewer than 19, orfewer than 18, or fewer than 17, or fewer than 16, or fewer than 15matches out of 22 contiguous nucleotides of a non-target gene sequence.

In some embodiments, it may be desirable to design the recombinant DNAconstruct to include regions predicted to not generate undesirablepolypeptides, for example, by screening the recombinant DNA constructfor sequences that may encode known undesirable polypeptides or closehomologues of these. Undesirable polypeptides include, but are notlimited to, polypeptides homologous to known allergenic polypeptides andpolypeptides homologous to known polypeptide toxins. Publicly availablesequences encoding such undesirable potentially allergenic peptides areavailable, for example, the Food Allergy Research and Resource Program(FARRP) allergen database (available at allergenonline.com) or theBiotechnology Information for Food Safety Databases (available atwww.iit.edu/˜sgendel/fa.htm) (see also, for example, Gendel (1998) Adv.Food Nutr. Res., 42:63-92). Undesirable sequences can also include, forexample, those polypeptide sequences annotated as known toxins or aspotential or known allergens and contained in publicly availabledatabases such as GenBank, EMBL, SwissProt, and others, which aresearchable by the Entrez system (www.ncbi.nih.gov/Entrez). Non-limitingexamples of undesirable, potentially allergenic peptide sequencesinclude glycinin from soybean, oleosin and agglutinin from peanut,glutenins from wheat, casein, lactalbumin, and lactoglobulin from bovinemilk, and tropomyosin from various shellfish (allergenonline.com).Non-limiting examples of undesirable, potentially toxic peptides includetetanus toxin tetA from Clostridium tetani, diarrheal toxins fromStaphylococcus aureus, and venoms such as conotoxins from Conus spp. andneurotoxins from arthropods and reptiles (www.ncbi.nih.gov/Entrez).

In one non-limiting example, the recombinant DNA construct is screenedto eliminate those transcribable sequences encoding polypeptides withperfect homology to a known allergen or toxin over 8 contiguous aminoacids, or with at least 35% identity over at least 80 amino acids; suchscreens can be performed on any and all possible reading frames in bothdirections, on potential open reading frames that begin with AUG (ATG inthe corresponding DNA), or on all possible reading frames, regardless ofwhether they start with an AUG (or ATG) or not. When a “hit” or match ismade, that is, when a sequence that encodes a potential polypeptide withperfect homology to a known allergen or toxin over 8 contiguous aminoacids (or at least about 35% identity over at least about 80 aminoacids), is identified, the nucleic acid sequences corresponding to thehit can be avoided, eliminated, or modified when selecting sequences tobe used in an RNA for silencing a target gene. In one embodiment therecombinant DNA construct is designed so no potential open reading framethat begins with AUG (ATG in the corresponding DNA) is included.Avoiding, elimination of, or modification of, an undesired sequence canbe achieved by any of a number of methods known to those skilled in theart. In some cases, the result can be novel sequences that are believedto not exist naturally. For example, avoiding certain sequences can beaccomplished by joining together “clean” sequences into novel chimericsequences to be used in the recombinant DNA construct.

Applicants recognize that in some microRNA-mediated gene silencing, itis possible for imperfectly matching miRNA sequences to be effective atgene silencing. For example, it has been shown that mismatches near thecenter of a miRNA complementary site has stronger effects on the miRNA'sgene silencing than do more distally located mismatches. See, forexample, FIG. 4 in Mallory et al. (2004) EMBO J., 23:3356-3364. Inanother example, it has been reported that, both the position of amismatched base pair and the identity of the nucleotides forming themismatch influence the ability of a given siRNA to silence a targetgene, and that adenine-cytosine mismatches, in addition to the G:Uwobble base pair, were well tolerated (see Du et al. (2005) NucleicAcids Res., 33:1671-1677). Thus, a given strand of the recombinant DNAconstruct need not always have 100% sequence identity with the intendedtarget gene, but generally would preferably have substantial sequenceidentity with the intended target gene, such as about 95%, about 90%,about 85%, or about 80% sequence identity with the intended target gene.Described in terms of complementarity, one strand of the recombinant DNAconstruct is preferably designed to have substantial complementarity tothe intended target (e.g., a target messenger RNA or target non-codingRNA), such as about 95%, about 90%, about 85%, or about 80%complementarity to the intended target. In a non-limiting example, inthe case of a recombinant DNA construct encoding a mature miRNA of 22nucleotides, the encoded mature miRNA is designed to be is substantiallybut not perfectly complementary to 22 contiguous nucleotides of a targetRNA; preferably the nucleotide at position 22 is unpaired with thecorresponding position in the target RNA to prevent transitivity.

Persons of ordinary skill in the art are capable of judging theimportance given to screening for regions predicted to be more highlyspecific to the target gene or predicted to not generate undesirablepolypeptides, relative to the importance given to other criteria, suchas, but not limited to, the percent sequence identity with the intendedtarget gene or the predicted gene silencing efficiency of a givensequence. For example, a recombinant DNA construct of this invention isdesigned to be processed to a mature miRNA that is active across severaltarget invertebrate pest species, and therefore one skilled in the artcan determine that it is more important to include in the recombinantDNA construct DNA encoding a mature miRNA that is specific to theseveral invertebrate pest species of interest, but less important toscreen for regions predicted to have higher gene silencing efficiency orfor regions predicted to generate undesirable polypeptides.

Promoters

Generally, the recombinant DNA construct of this invention includes apromoter, functional in a plant cell, and operably linked to the DNAencoding the recombinant miRNA precursor. In various embodiments, thepromoter is selected from the group consisting of a constitutivepromoter, a spatially specific promoter, a temporally specific promoter,a developmentally specific promoter, and an inducible promoter.

Non-constitutive promoters suitable for use with the recombinant DNAconstructs of the invention include spatially specific promoters,temporally specific promoters, and inducible promoters. Spatiallyspecific promoters can include organelle-, cell-, tissue-, ororgan-specific promoters (e.g., a plastid-specific, a root-specific, apollen-specific, or a seed-specific promoter for suppressing expressionof the first target RNA in plastids, roots, pollen, or seeds,respectively). In many cases a seed-specific, embryo-specific,aleurone-specific, or endosperm-specific promoter is especially useful.Temporally specific promoters can include promoters that tend to promoteexpression during certain developmental stages in a plant's growthcycle, or during different times of day or night, or at differentseasons in a year. Inducible promoters include promoters induced bychemicals or by environmental conditions such as, but not limited to,biotic or abiotic stress (e.g., water deficit or drought, heat, cold,high or low nutrient or salt levels, high or low light levels, or pestor pathogen infection). Of particular interest are microRNA promoters,especially those having a temporally specific, spatially specific, orinducible expression pattern. An expression-specific promoter can alsoinclude promoters that are generally constitutively expressed but atdiffering degrees or “strengths” of expression, including promoterscommonly regarded as “strong promoters” or as “weak promoters”.

Promoters of particular interest include the following non-limitingexamples: an opaline synthase promoter isolated from T-DNA ofAgrobacterium; a cauliflower mosaic virus 35S promoter; enhancedpromoter elements or chimeric promoter elements such as an enhancedcauliflower mosaic virus (CaMV) 35S promoter linked to an enhancerelement (an intron from heat shock protein 70 of Zea mays); rootspecific promoters such as those disclosed in U.S. Pat. Nos. 5,837,848;6,437,217 and 6,426,446; a maize L3 oleosin promoter disclosed in U.S.Pat. No. 6,433,252; a promoter for a plant nuclear gene encoding aplastid-localized aldolase disclosed in U. S. Patent ApplicationPublication 2004/0216189; cold-inducible promoters disclosed in U.S.Pat. No. 6,084,089; salt-inducible promoters disclosed in U.S. Pat. No.6,140,078; light-inducible promoters disclosed in U.S. Pat. No.6,294,714; pathogen-inducible promoters disclosed in U.S. Pat. No.6,252,138; and water deficit-inducible promoters disclosed in U.S.Patent Application Publication 2004/0123347 A1. All of theabove-described patents and patent publications disclosing promoters andtheir use, especially in recombinant DNA constructs functional in plantsare incorporated herein by reference.

Plant vascular- or phloem-specific promoters of interest include a rolCor rolA promoter of Agrobacterium rhizogenes, a promoter of aAgrobacterium tumefaciens T-DNA gene 5, the rice sucrose synthase RSs1gene promoter, a Commelina yellow mottle badnavirus promoter, a coconutfoliar decay virus promoter, a rice tungro bacilliform virus promoter,the promoter of a pea glutamine synthase GS3A gene, a invCD111 andinvCD141 promoters of a potato invertase genes, a promoter isolated fromArabidopsis shown to have phloem-specific expression in tobacco byKertbundit et al. (1991), a VAHOX1 promoter region, a pea cell wallinvertase gene promoter, an acid invertase gene promoter from carrot, apromoter of a sulfate transporter gene Sultr1;3, a promoter of a plantsucrose synthase gene, and a promoter of a plant sucrose transportergene.

The promoter element can include nucleic acid sequences that are notnaturally occurring promoters or promoter elements or homologues thereofbut that can regulate expression of a gene. Examples of such “geneindependent” regulatory sequences include naturally occurring orartificially designed RNA sequences that include a ligand-binding regionor aptamer and a regulatory region (which can be cis-acting). See, forexample, Isaacs et al. (2004) Nat. Biotechnol., 22:841-847, Bayer andSmolke (2005) Nature Biotechnol., 23:337-343, Mandal and Breaker (2004)Nature Rev. Mol. Cell Biol., 5:451-463, Davidson and Ellington (2005)Trends Biotechnol., 23:109-112, Winkler et al. (2002) Nature,419:952-956, Sudarsan et al. (2003) RNA, 9:644-647, and Mandal andBreaker (2004) Nature Struct. Mol. Biol., 11:29-35. Such“riboregulators” could be selected or designed for specific spatial ortemporal specificity, for example, to regulate translation of the DNAencoding the recombinant miRNA precursor only in the presence (orabsence) of a given concentration of the appropriate ligand. Onenon-limiting example is a riboregulator that is responsive to anendogenous ligand (e.g., jasmonic acid or salicylic acid) produced bythe plant when under stress (e.g., abiotic stress such as water,temperature, or nutrient stress, or biotic stress such as attach bypests or pathogens); under stress, the level of endogenous ligandincreases to a level sufficient for the riboregulator to begintranscription of the DNA encoding the recombinant miRNA precursor.

Making and Using Recombinant DNA Constructs

The recombinant DNA constructs of this invention are made by any methodsuitable to the intended application, taking into account, for example,the type of expression desired and convenience of use in the plant inwhich the construct is to be transcribed. General methods for making andusing DNA constructs and vectors are well known in the art and describedin detail in, for example, handbooks and laboratory manuals includingSambrook and Russell, “Molecular Cloning: A Laboratory Manual” (thirdedition), Cold Spring Harbor Laboratory Press, NY, 2001. An example ofuseful technology for building DNA constructs and vectors fortransformation is disclosed in U. S. Patent Application Publication2004/0115642 A1, incorporated herein by reference. DNA constructs canalso be built using the GATEWAY™ cloning technology (available fromInvitrogen Life Technologies, Carlsbad, Calif.), which uses thesite-specific recombinase LR cloning reaction of the Integrase/attsystem from bacteriophage lambda vector construction, instead ofrestriction endonucleases and ligases. The LR cloning reaction isdisclosed in U.S. Pat. Nos. 5,888,732 and 6,277,608, and in U. S. PatentApplication Publications 2001/283529, 2001/282319 and 2002/0007051, allof which are incorporated herein by reference. The GATEWAY™ CloningTechnology Instruction Manual, which is also supplied by Invitrogen,provides concise directions for routine cloning of any desired DNA intoa vector comprising operable plant expression elements. Anotheralternative vector fabrication method employs ligation-independentcloning as disclosed by Aslandis et al. (1990) Nucleic Acids Res.,18:6069-6074 and Rashtchian et al. (1992) Biochem., 206:91-97, where aDNA fragment with single-stranded 5′ and 3′ ends is ligated into adesired vector which can then be amplified in vivo.

In certain embodiments, the DNA sequence of the recombinant DNAconstruct includes sequence that has been codon-optimized for the plantin which the recombinant DNA construct is to be expressed. For example,a recombinant DNA construct to be expressed in a plant can have all orparts of its sequence (e.g., the first gene suppression element or thegene expression element) codon-optimized for expression in a plant bymethods known in the art. See, e.g., U.S. Pat. No. 5,500,365,incorporated by reference, for a description of codon-optimizationmethodology for plants; see also De Amicis and Marchetti (2000) NucleicAcid Res., 28:3339-3346.

Transgeninc Plant Cells and Plants

Another aspect of this invention provides a non-natural transgenic plantcell having in its genome recombinant DNA that is transcribed in thenon-natural transgenic plant cell to a recombinant miRNA precursor,wherein the recombinant miRNA precursor includes a single strand of RNAthat folds into the secondary structure of an invertebrate miRNAprecursor and that includes at least one stem-loop that is processed toa mature miRNA, and wherein the mature miRNA suppresses expression of atleast one target gene of an invertebrate or of a symbiont associatedwith the invertebrate. Also provided are a non-natural transgenic plantcontaining the non-natural transgenic plant cell of this invention, anon-natural transgenic plant grown from the non-natural transgenic plantcell of this invention, and non-natural transgenic seed produced by thenon-natural transgenic plants. Such non-natural transgenic plant cells,plants, and seeds can be made using the techniques described below underthe heading “Making and Using Transgenic Plant Cells and TransgenicPlants”. This invention further provides a method of suppressing atleast one target gene of an invertebrate pest of a plant or of asymbiont associated with the invertebrate, including providing a plantincluding the non-natural transgenic plant cell of this invention,wherein the invertebrate is the invertebrate pest, the recombinant DNAis transcribed in the non-natural transgenic plant cell to therecombinant miRNA precursor, and when the invertebrate pest ingests therecombinant miRNA precursor, the at least one target gene is suppressed.

The non-natural transgenic plant of this invention includes plants ofany developmental stage, and includes a non-natural regenerated plantprepared from the non-natural transgenic plant cells disclosed herein,or a non-natural progeny plant (which can be an inbred or hybrid progenyplant) of the regenerated plant, or seed of such a non-naturaltransgenic plant. Also provided and claimed is a non-natural transgenicseed having in its genome a recombinant DNA construct of this invention.The non-natural transgenic plant cells, transgenic plants, andtransgenic seeds of this invention are made by methods well-known in theart, as described below under the heading “Making and Using TransgenicPlant Cells and Transgenic Plants”.

The non-natural transgenic plant cell can include an isolated plant cell(e.g., individual plant cells or cells grown in or on an artificialculture medium), or can include a plant cell in undifferentiated tissue(e.g., callus or any aggregation of plant cells). The non-naturaltransgenic plant cell can include a plant cell in at least onedifferentiated tissue selected from the group consisting of leaf (e.g.,petiole and blade), root, stem (e.g., tuber, rhizome, stolon, bulb, andcorm) stalk (e.g., xylem, phloem), wood, seed, fruit (e.g., nut, grain,fleshy fruits), and flower (e.g., stamen, filament, anther, pollen,carpel, pistil, ovary, ovules).

The non-natural transgenic plant cell or non-natural transgenic plant ofthe invention can be any suitable plant cell or plant of interest. Bothtransiently transformed and stably transformed plant cells areencompassed by this invention. Stably transformed transgenic plants areparticularly preferred. In many preferred embodiments, the non-naturaltransgenic plant is a fertile transgenic plant from which seed can beharvested, and the invention further claims non-natural transgenic seedof such transgenic plants, wherein the non-natural seed preferably alsocontains the recombinant construct of this invention.

Making and Using Transgenic Plant Cells and Transgenic Plants

Where a recombinant DNA construct of this invention is used to produce anon-natural transgenic plant cell, transgenic plant, or transgenic seedof this invention, transformation can include any of the well-known anddemonstrated methods and compositions. Suitable methods for planttransformation include virtually any method by which DNA can beintroduced into a cell, such as by direct delivery of DNA (e.g., byPEG-mediated transformation of protoplasts, by electroporation, byagitation with silicon carbide fibers, and by acceleration of DNA coatedparticles), by Agrobacterium-mediated transformation, by viral or othervectors, etc. One preferred method of plant transformation ismicroprojectile bombardment, for example, as illustrated in U.S. Pat.No. 5,015,580 (soy), U.S. Pat. No. 5,550,318 (maize), U.S. Pat. No.5,538,880 (maize), U.S. Pat. No. 6,153,812 (wheat), U.S. Pat. No.6,160,208 (maize), U.S. Pat. No. 6,288,312 (rice) and U.S. Pat. No.6,399,861 (maize), and U.S. Pat. No. 6,403,865 (maize), all of which areincorporated by reference.

Another preferred method of plant transformation isAgrobacterium-mediated transformation. In one preferred embodiment, thetransgenic plant cell of this invention is obtained by transformation bymeans of Agrobacterium containing a binary Ti plasmid system, whereinthe Agrobacterium carries a first Ti plasmid and a second, chimericplasmid containing at least one T-DNA border of a wild-type Ti plasmid,a promoter functional in the transformed plant cell and operably linkedto a gene suppression construct of the invention. See, for example, thebinary system described in U.S. Pat. No. 5,159,135, incorporated byreference. Also see De Framond (1983) Biotechnology, 1:262-269; andHoekema et al., (1983) Nature, 303:179. In such abinary system, thesmaller plasmid, containing the T-DNA border or borders, can beconveniently constructed and manipulated in a suitable alternative host,such as E. coli, and then transferred into Agrobacterium.

Detailed procedures for Agrobacterium-mediated transformation of plants,especially crop plants, include, for example, procedures disclosed inU.S. Pat. Nos. 5,004,863, 5,159,135, and 5,518,908 (cotton); U.S. Pat.Nos. 5,416,011, 5,569,834, 5,824,877 and 6,384,301 (soy); U.S. Pat. Nos.5,591,616 and 5,981,840 (maize); U.S. Pat. No. 5,463,174 (brassicas),and in U. S. Patent Application Publication 2004/0244075 (maize), all ofwhich are incorporated by reference. Similar methods have been reportedfor many plant species, both dicots and monocots, including, amongothers, peanut (Cheng et al. (1996) Plant Cell Rep., 15: 653); asparagus(Bytebier et al. (1987) Proc. Natl. Acad. Sci. U.S.A., 84:5345); barley(Wan and Lemaux (1994) Plant Physiol., 104:37); rice (Toriyama et al(1988) Bio/Technology, 6:10; Zhang et al (1988) Plant Cell Rep., 7:379;wheat (Vasil et al. (1992) Bio/Technology, 10:667; Becker et al. (1994)Plant J., 5:299), alfalfa (Masoud et al (1996) Transgen. Res., 5:313);and tomato (Sun et al. (2006) Plant Cell Physiol., 47:426-431). See alsoa description of vectors, transformation methods, and production oftransformed Arabidopsis thaliana plants where transcription factors areconstitutively expressed by a CaMV35S promoter, in U. S. PatentApplication Publication 2003/0167537 A1, incorporated by reference.Transgenic plant cells and transgenic plants can also be obtained bytransformation with other vectors, such as, but not limited to, viralvectors (e.g., tobacco etch potyvirus (TEV), barley stripe mosaic virus(BSMV), and the viruses referenced in Edwardson and Christie, “ThePotyvirus Group: Monograph No. 16, 1991, Agric. Exp. Station, Univ. ofFlorida), plasmids, cosmids, YACs (yeast artificial chromosomes), BACs(bacterial artificial chromosomes) or any other suitable cloning vector,when used with an appropriate transformation protocol, e.g., bacterialinfection (e.g., with Agrobacterium as described above), binarybacterial artificial chromosome constructs, direct delivery of DNA(e.g., via PEG-mediated transformation, desiccation/inhibition-mediatedDNA uptake, electroporation, agitation with silicon carbide fibers, andmicroprojectile bombardment). It would be clear to one of ordinary skillin the art that various transformation methodologies can be used andmodified for production of stable transgenic plants from any number ofplant species of interest.

Transformation methods to provide transgenic plant cells and transgenicplants containing stably integrated recombinant DNA are preferablypracticed in tissue culture on media and in a controlled environment.“Media” refers to the numerous nutrient mixtures that are used to growcells in vitro, that is, outside of the intact living organism.Recipient cell targets include, but are not limited to, meristem cells,callus, immature embryos or parts of embryos, and gametic cells such asmicrospores, pollen, sperm, and egg cells. Any cell from which a fertileplant can be regenerated is contemplated as a useful recipient cell forpractice of the invention. Callus can be initiated from various tissuesources, including, but not limited to, immature embryos or parts ofembryos, seedling apical meristems, microspores, and the like. Thosecells which are capable of proliferating as callus can serve asrecipient cells for genetic transformation. Practical transformationmethods and materials for making transgenic plants of this invention(e.g., various media and recipient target cells, transformation ofimmature embryos, and subsequent regeneration of fertile transgenicplants) are disclosed, for example, in U.S. Pat. Nos. 6,194,636 and6,232,526 and U. S. Patent Application Publication 2004/0216189, whichare incorporated by reference.

In general transformation practice, DNA is introduced into only a smallpercentage of target cells in any one transformation experiment. Markergenes are generally used to provide an efficient system foridentification of those cells that are stably transformed by receivingand integrating a transgenic DNA construct into their genomes. Preferredmarker genes provide selective markers which confer resistance to aselective agent, such as an antibiotic or herbicide. Any of theantibiotics or herbicides to which a plant cell may be resistant can bea useful agent for selection. Potentially transformed cells are exposedto the selective agent. In the population of surviving cells will bethose cells where, generally, the resistance-conferring gene isintegrated and expressed at sufficient levels to permit cell survival.Cells can be tested further to confirm stable integration of therecombinant DNA. Commonly used selective marker genes include thoseconferring resistance to antibiotics such as kanamycin or paromomycin(nptII), hygromycin B (aph IV) and gentamycin (aac3 and aacC4) orresistance to herbicides such as glufosinate (bar or pat) and glyphosate(EPSPS). Examples of useful selective marker genes and selection agentsare illustrated in U.S. Pat. Nos. 5,550,318, 5,633,435, 5,780,708, and6,118,047, all of which are incorporated by reference. Screenablemarkers or reporters, such as markers that provide an ability tovisually identify transformants can also be employed. Non-limitingexamples of useful screenable markers include, for example, a geneexpressing a protein that produces a detectable color by acting on achromogenic substrate (e.g., beta-glucuronidase (GUS) (uidA) orluciferase (luc)) or that itself is detectable, such as greenfluorescent protein (GFP) (gfp) or an immunogenic molecule. Those ofskill in the art will recognize that many other useful markers orreporters are available for use.

Detecting or measuring transcription of the recombinant DNA construct inthe transgenic plant cell of the invention can be achieved by anysuitable method, including protein detection methods (e.g., westernblots, ELISAs, and other immunochemical methods), measurements ofenzymatic activity, or nucleic acid detection methods (e.g., Southernblots, northern blots, PCR, RT-PCR, fluorescent in situ hybridization).Such methods are well known to those of ordinary skill in the art asevidenced by the numerous handbooks available; see, for example, JosephSambrook and David W. Russell, “Molecular Cloning: A Laboratory Manual”(third edition), Cold Spring Harbor Laboratory Press, NY, 2001;Frederick M. Ausubel et al. (editors) “Short Protocols in MolecularBiology” (fifth edition), John Wiley and Sons, 2002; John M. Walker(editor) “Protein Protocols Handbook” (second edition), Humana Press,2002; and Leandro Pena (editor) “Transgenic Plants: Methods andProtocols”, Humana Press, 2004.

Other suitable methods for detecting or measuring transcription of therecombinant DNA construct in the transgenic plant cell of the inventioninclude measurement of any other trait that is a direct or proxyindication of suppression of the target gene in the transgenic plantcell in which the recombinant DNA construct is transcribed, relative toone in which the recombinant DNA is not transcribed, e.g., gross ormicroscopic morphological traits, growth rates, yield, reproductive orrecruitment rates, resistance to pests or pathogens, or resistance tobiotic or abiotic stress (e.g., water deficit stress, salt stress,nutrient stress, heat or cold stress). Such methods can use directmeasurements of a phenotypic trait or proxy assays (e.g., in plants,these assays include plant part assays such as leaf or root assays todetermine tolerance of abiotic stress). Non-limiting methods includedirect measurements of resistance to the invertebrate pest (e.g., damageto plant tissues) or proxy assays (e.g., plant yield assays, orbioassays such as the Western corn rootworm (Diabrotica virgiferavirgifera LeConte) larval bioassay described in International PatentApplication Publication WO2005/110068 A2 and U. S. Patent ApplicationPublication US 2006/0021087 A1, incorporated by reference, or thesoybean cyst nematode bioassay described by Steeves et al. (2006) Funct.Plant Biol., 33:991-999, wherein cysts per plant, cysts per gram root,eggs per plant, eggs per gram root, and eggs per cyst are measured.

The recombinant DNA constructs of the invention can be stacked withother recombinant DNA for imparting additional traits (e.g., in the caseof transformed plants, traits including herbicide resistance, pestresistance, cold germination tolerance, water deficit tolerance, and thelike) for example, by expressing or suppressing other genes. Constructsfor coordinated decrease and increase of gene expression are disclosedin U.S. Patent Application Publication 2004/0126845 A1, incorporated byreference.

Seeds of transgenic, fertile plants can be harvested and used to growprogeny generations, including hybrid generations, of transgenic plantsof this invention that include the recombinant DNA construct in theirgenome. Thus, in addition to direct transformation of a plant with arecombinant DNA construct of this invention, transgenic plants of theinvention can be prepared by crossing a first plant having therecombinant DNA with a second plant lacking the construct. For example,the recombinant DNA can be introduced into a plant line that is amenableto transformation to produce a transgenic plant, which can be crossedwith a second plant line to introgress the recombinant DNA into theresulting progeny. A transgenic plant of the invention can be crossedwith a plant line having other recombinant DNA that confers one or moreadditional trait(s) (such as, but not limited to, herbicide resistance,pest or disease resistance, environmental stress resistance, modifiednutrient content, and yield improvement) to produce progeny plantshaving recombinant DNA that confers both the desired target sequenceexpression behavior and the additional trait(s).

Typically, in such breeding for combining traits the transgenic plantdonating the additional trait is a male line and the transgenic plantcarrying the base traits is the female line. The progeny of this crosssegregate such that some of the plant will carry the DNA for bothparental traits and some will carry DNA for one parental trait; suchplants can be identified by markers associated with parental recombinantDNA Progeny plants carrying DNA for both parental traits can be crossedback into the female parent line multiple times, e.g., usually 6 to 8generations, to produce a progeny plant with substantially the samegenotype as one original transgenic parental line but for therecombinant DNA of the other transgenic parental line.

Yet another aspect of the invention is a transgenic plant grown from thetransgenic seed of the invention. This invention contemplates transgenicplants grown directly from transgenic seed containing the recombinantDNA as well as progeny generations of plants, including inbred or hybridplant lines, made by crossing a transgenic plant grown directly fromtransgenic seed to a second plant not grown from the same transgenicseed.

Crossing can include, for example, the following steps:

-   -   (a) plant seeds of the first parent plant (e.g., non-transgenic        or a transgenic) and a second parent plant that is transgenic        according to the invention;    -   (b) grow the seeds of the first and second parent plants into        plants that bear flowers;    -   (c) pollinate a flower from the first parent with pollen from        the second parent; and    -   (d) harvest seeds produced on the parent plant bearing the        fertilized flower.

It is often desirable to introgress recombinant DNA into elitevarieties, e.g., by backcrossing, to transfer a specific desirable traitfrom one source to an inbred or other plant that lacks that trait. Thiscan be accomplished, for example, by first crossing a superior inbred(“A”) (recurrent parent) to a donor inbred (“B”) (non-recurrent parent),which carries the appropriate gene(s) for the trait in question, forexample, a construct prepared in accordance with the current invention.The progeny of this cross first are selected in the resultant progenyfor the desired trait to be transferred from the non-recurrent parent“B”, and then the selected progeny are mated back to the superiorrecurrent parent “A”. After five or more backcross generations withselection for the desired trait, the progeny are hemizygous for locicontrolling the characteristic being transferred, but are like thesuperior parent for most or almost all other genes. The last backcrossgeneration would be selfed to give progeny which are pure breeding forthe gene(s) being transferred, i.e., one or more transformation events.

Through a series of breeding manipulations, a selected DNA construct canbe moved from one line into an entirely different line without the needfor further recombinant manipulation. One can thus produce inbred plantswhich are true breeding for one or more DNA constructs. By crossingdifferent inbred plants, one can produce a large number of differenthybrids with different combinations of DNA constructs. In this way,plants can be produced which have the desirable agronomic propertiesfrequently associated with hybrids (“hybrid vigor”), as well as thedesirable characteristics imparted by one or more DNA constructs.

Genetic markers can be used to assist in the introgression of one ormore DNA constructs of the invention from one genetic background intoanother. Marker assisted selection offers advantages relative toconventional breeding in that it can be used to avoid errors caused byphenotypic variations. Further, genetic markers can provide dataregarding the relative degree of elite germplasm in the individualprogeny of a particular cross. For example, when a plant with a desiredtrait which otherwise has a non-agronomically desirable geneticbackground is crossed to an elite parent, genetic markers can be used toselect progeny which not only possess the trait of interest, but alsohave a relatively large proportion of the desired germplasm. In thisway, the number of generations required to introgress one or more traitsinto a particular genetic background is minimized. The usefulness ofmarker assisted selection in breeding transgenic plants of the currentinvention, as well as types of useful molecular markers, such as but notlimited to SSRs and SNPs, are discussed in PCT Application PublicationWO 02/062129 and U. S. Patent Application Publications Numbers2002/0133852, 2003/0049612, and 2003/0005491, each of which isincorporated by reference in their entirety.

In certain transgenic plant cells and transgenic plants of theinvention, it may be desirable to concurrently express (or suppress) agene of interest while also regulating expression of a target gene.Thus, in some embodiments, the transgenic plant contains recombinant DNAfurther including a gene expression (or suppression) element forexpressing at least one gene of interest, and transcription of therecombinant miRNA precursor is preferably effected with concurrenttranscription of the gene expression (or suppression) element.

Thus, as described herein, the non-natural transgenic plant cells ortransgenic plants of the invention can be obtained by use of anyappropriate transient or stable, integrative or non-integrativetransformation method known in the art or presently disclosed. Therecombinant DNA constructs can be transcribed in any plant cell ortissue or in a whole plant of any developmental stage. Transgenic plantscan be derived from any monocot or dicot plant, such as, but not limitedto, plants of commercial or agricultural interest, such as crop plants(especially crop plants used for human food or animal feed), wood- orpulp-producing trees, vegetable plants, fruit plants, and ornamentalplants. Non-limiting examples of plants of interest include grain cropplants (such as wheat, oat, barley, maize, rye, triticale, rice, millet,sorghum, quinoa, amaranth, and buckwheat); forage crop plants (such asforage grasses and forage dicots including alfalfa, vetch, clover, andthe like); oilseed crop plants (such as cotton, safflower, sunflower,soybean, canola, rapeseed, flax, peanuts, and oil palm); tree nuts (suchas walnut, cashew, hazelnut, pecan, almond, and the like); sugarcane,coconut, date palm, olive, sugarbeet, tea, and coffee; wood- orpulp-producing trees; vegetable crop plants such as legumes (forexample, beans, peas, lentils, alfalfa, peanut), lettuce, asparagus,artichoke, celery, carrot, radish, the brassicas (for example, cabbages,kales, mustards, and other leafy brassicas, broccoli, cauliflower,Brussels sprouts, turnip, kohlrabi), edible cucurbits (for example,cucumbers, melons, summer squashes, winter squashes), edible alliums(for example, onions, garlic, leeks, shallots, chives), edible membersof the Solanaceae (for example, tomatoes, eggplants, potatoes, peppers,groundcherries), and edible members of the Chenopodiaceae (for example,beet, chard, spinach, quinoa, amaranth); fruit crop plants such asapple, pear, citrus fruits (for example, orange, lime, lemon,grapefruit, and others), stone fruits (for example, apricot, peach,plum, nectarine), banana, pineapple, grape, kiwifruit, papaya, avocado,and berries; and ornamental plants including ornamental floweringplants, ornamental trees and shrubs, ornamental groundcovers, andornamental grasses. Preferred dicot plants include, but are not limitedto, canola, broccoli, cabbage, carrot, cauliflower, Chinese cabbage,cucumber, dry beans, eggplant, fennel, garden beans, gourds, lettuces,melons, okra, peas, peppers, pumpkin, radishes, spinach, squash,watermelon, cotton, potato, quinoa, amaranth, buckwheat, safflower,soybean, sugarbeet, and sunflower. Preferred monocots include, but arenot limited to, wheat, oat, barley, maize (including sweet corn andother varieties), rye, triticale, rice, ornamental and forage grasses,sorghum, millet, onions, leeks, and sugarcane, more preferably maize,wheat, and rice.

The ultimate goal in plant transformation is to produce plants which areuseful to man. In this respect, non-natural transgenic plants of theinvention can be used for virtually any purpose deemed of value to thegrower or to the consumer. For example, one may wish to harvest thetransgenic plant itself, or harvest transgenic seed of the transgenicplant for planting purposes, or products can be made from the transgenicplant or its seed such as oil, starch, ethanol or other fermentationproducts, animal feed or human food, pharmaceuticals, and variousindustrial products. For example, maize is used extensively in the foodand feed industries, as well as in industrial applications. Furtherdiscussion of the uses of maize can be found, for example, in U.S. Pat.Nos. 6,194,636, 6,207,879, 6,232,526, 6,426,446, 6,429,357, 6,433,252,6,437,217, and 6,583,338, incorporated by reference, and PCTPublications WO 95/06128 and WO 02/057471. Thus, this invention alsoprovides commodity products produced from a non-natural transgenic plantcell, plant, or seed of this invention, including, but not limited to,harvested leaves, roots, shoots, tubers, stems, fruits, seeds, or otherparts of a plant, meals, oils, extracts, fermentation or digestionproducts, crushed or whole grains or seeds of a plant, or any food ornon-food product including such commodity products produced from atransgenic plant cell, plant, or seed of this invention. The detectionof one or more of nucleic acid sequences of the recombinant DNAconstructs of this invention in one or more commodity or commodityproducts contemplated herein is de facto evidence that the commodity orcommodity product contains or is derived from a non-natural transgenicplant cell, plant, or seed of this invention.

In preferred embodiments, the non-natural transgenic plant prepared fromthe non-natural transgenic plant cell of this invention, i.e., anon-natural transgenic plant having in its genome a recombinant DNAconstruct of this invention has at least one additional altered trait,relative to a plant lacking the recombinant DNA construct, selected fromthe group of traits consisting of:

-   -   (a) improved abiotic stress tolerance;    -   (b) improved biotic stress tolerance;    -   (c) modified primary metabolite composition;    -   (d) modified secondary metabolite composition;    -   (e) modified trace element, carotenoid, or vitamin composition;    -   (f) improved yield;    -   (g) improved ability to use nitrogen or other nutrients;    -   (h) modified agronomic characteristics;    -   (i) modified growth or reproductive characteristics; and    -   (j) improved harvest, storage, or processing quality.

In particularly preferred embodiments, the non-natural transgenic plantis characterized by: improved tolerance of abiotic stress (e.g.,tolerance of water deficit or drought, heat, cold, non-optimal nutrientor salt levels, non-optimal light levels) or of biotic stress (e.g.,crowding, allelopathy, or wounding); by a modified primary metabolite(e.g., fatty acid, oil, amino acid, protein, sugar, or carbohydrate)composition; a modified secondary metabolite (e.g., alkaloids,terpenoids, polyketides, non-ribosomal peptides, and secondarymetabolites of mixed biosynthetic origin) composition; a modified traceelement (e.g., iron, zinc), carotenoid (e.g., beta-carotene, lycopene,lutein, zeaxanthin, or other carotenoids and xanthophylls), or vitamin(e.g., tocopherols) composition; improved yield (e.g., improved yieldunder non-stress conditions or improved yield under biotic or abioticstress); improved ability to use nitrogen or other nutrients; modifiedagronomic characteristics (e.g., delayed ripening; delayed senescence;earlier or later maturity; improved shade tolerance; improved resistanceto root or stalk lodging; improved resistance to “green snap” of stems;modified photoperiod response); modified growth or reproductivecharacteristics (e.g., intentional dwarfing; intentional male sterility,useful, e.g., in improved hybridization procedures; improved vegetativegrowth rate; improved germination; improved male or female fertility);improved harvest, storage, or processing quality (e.g., improvedresistance to pests during storage, improved resistance to breakage,improved appeal to consumers); or any combination of these traits.

In one preferred embodiment, non-natural transgenic seed, or seedproduced by the non-natural transgenic plant, has modified primarymetabolite (e.g., fatty acid, oil, amino acid, protein, sugar, orcarbohydrate) composition, a modified secondary metabolite (e.g.,alkaloids, terpenoids, polyketides, non-ribosomal peptides, andsecondary metabolites of mixed biosynthetic origin) composition, amodified trace element (e.g., iron, zinc), carotenoid (e.g.,beta-carotene, lycopene, lutein, zeaxanthin, or other carotenoids andxanthophylls), or vitamin (e.g., tocopherols,) composition, an improvedharvest, storage, or processing quality, or a combination of these. Forexample, it can be desirable to modify the amino acid (e.g., lysine,methionine, tryptophan, or total protein), oil (e.g., fatty acidcomposition or total oil), carbohydrate (e.g., simple sugars orstarches), trace element, carotenoid, or vitamin content of seeds ofcrop plants (e.g., canola, cotton, safflower, soybean, sugarbeet,sunflower, wheat, maize, or rice), preferably in combination withimproved seed harvest, storage, or processing quality, and thus provideimproved seed for use in animal feeds or human foods. In anotherinstance, it can be desirable to change levels of native components ofthe transgenic plant or seed of a transgenic plant, for example, todecrease levels of proteins with low levels of lysine, methionine, ortryptophan, or to increase the levels of a desired amino acid or fattyacid, or to decrease levels of an allergenic protein or glycoprotein(e.g., peanut allergens including ara h 1, wheat allergens includinggliadins and glutenins, soy allergens including P34 allergen, globulins,glycinins, and conglycinins) or of a toxic metabolite (e.g., cyanogenicglycosides in cassava, solanum alkaloids in members of the Solanaceae).

EXAMPLES Example 1

This example describes non-limiting embodiments of recombinant DNAconstructs useful in making the non-natural transgenic plant cells,plants, and seeds of this invention. More particularly, this exampledescribes a recombinant DNA construct that is transcribable in a plantcell to a recombinant miRNA precursor, wherein the recombinant miRNAprecursor includes a single strand of RNA that folds into the secondarystructure of an invertebrate miRNA precursor and that includes at leastone stem-loop that is processed to a mature miRNA; and wherein themature miRNA is designed to suppress expression of at least one targetgene of an invertebrate or of a symbiont associated with theinvertebrate. This construct transcribes to a recombinant miRNAprecursor that is relatively stable in planta, allowing ingestion of therelatively intact recombinant miRNA precursor by an invertebrate.

In one non-limiting embodiment of this invention, the recombinant DNAconstruct includes sequence derived from multiple miRNA precursors,e.g., a polycistronic cluster of 8 miRNAs (“8-mir”) identified onchromosome 2R in Drosophila melanogaster as reported by Biemar et al.(2005) Proc. Natl. Acad. Sci. U.S.A., 102:15907-15911. Using multiplemiRNA precursors (multiple different precursors, or multiple copies ofthe same precursor, or combinations thereof) results in multiple maturemiRNAs processed from a single recombinant DNA construct, which isadvantageous for targeting multiple target genes (e.g., differentalleles, different regions within a single target gene, or differenttarget genes) or increasing the amount of mature miRNA available forsilencing.

The “8-mir” cluster (SEQ ID NO. 1) is depicted in FIG. 1, with theindividual miRNA precursors indicated by bold underlined text. A shortersequence that includes all 8 miRNA precursors is also provided in SEQ IDNO. 2. This cluster includes DNA sequence encoding 8 miRNA precursors,arranged in this order: dme-mir-309 (SEQ ID NO. 3), dme-mir-3 (SEQ IDNO. 4), dme-mir-286 (SEQ ID NO. 5), dme-mir-4 (SEQ ID NO. 6), dme-mir-5(SEQ ID NO. 7), dme-mir-6-1 (SEQ ID NO. 8), dme-mir-6-2 (SEQ ID NO. 9),and dme-mir-6-3 (SEQ ID NO. 10). Table 1 identifies the DNA sequence andcorresponding RNA sequence for each miRNA precursor. The fold-backstructure (that is, the secondary structure of the miRNA precursorincluding a stem-loop that is processed to the mature miRNA) for each ofthe 8 miRNA precursors is depicted in FIG. 2, in which the mature miRNAis indicated within the fold-back structure in bold capitals.

TABLE 1 miRNA precursor DNA sequence RNA sequence dme-mir-309 SEQ ID NO.3 SEQ ID NO. 11 dme-mir-3 SEQ ID NO. 4 SEQ ID NO. 12 dme-mir-286 SEQ IDNO. 5 SEQ ID NO. 13 dme-mir-4 SEQ ID NO. 6 SEQ ID NO. 14 dme-mir-5 SEQID NO. 7 SEQ ID NO. 15 dme-mir-6-1 SEQ ID NO. 8 SEQ ID NO. 16dme-mir-6-2 SEQ ID NO. 9 SEQ ID NO. 17 dme-mir-6-3 SEQ ID NO. 10 SEQ IDNO. 18

In one embodiment, a recombinant DNA construct including a sequenceidentical to (or substantially similar to) the “8-miR” sequence (SEQ IDNO. 1) is expressed in a transgenic plant cell under the control of apromoter that differs from the native promoter of the native D.melanogaster “8-miR” cluster precursor. The target gene is theendogenous target of the mature miRNAs natively processed from the“8-miR” cluster, or a gene including sequence similar to the endogenoustarget (e.g., homologues or orthologues of the endogenous target).Techniques for predicting a target of a given animal miRNA are known inthe art and include those described by Lewis et al (2005) Cell,120:15-16, Lewis et al. (2003) Cell, 115:787-798, and Rehmsmeier et al.(2004) RNA, 10:1507-1517. A publicly available target predictor,TargetScanS (www.targetscan.org), predicts biological targets of miRNAsby searching for the presence of conserved 8mer and 7mer sites thatmatch the seed region (positions 2-7 of a mature miRNA) of each miRNA.Another publicly available on-line target predictor is RNAhybrid(bibiserv.techfak.uni-bielefeld.de/rnahybrid; see Krüger and Rehmsmeier(2006) Nucleic Acids Res., 34:W451-W454, and Rehmsmeier et al. (2004)RNA, 10:1507-1517). Also publicly available is an integrated database,miRGen (www.diana.pcbi.upenn.edu/miRGen; see Megraw et al (2007) NucleicAcids Res., 35:D149-D155) that provides (i) positional relationshipsbetween animal miRNAs and genomic annotation sets and (ii) animal miRNAtargets according to combinations of widely used target predictionprograms. Also publicly available is TarBase(www.diana.pcbi.upenn.edu/tarbase; see Sethupathy et al. (2006) RNA,12:192-197), a manually curated database of experimentally tested miRNAtargets, in human/mouse, fruit fly, worm, and zebrafish, whichdistinguishes between miRNA targets that have been validated (testedpositive) and those that tested negative.

In another embodiment, the “8-miR” sequence (SEQ ID NO. 1) serves as atemplate from which an “engineered 8-miR” sequence is derived, whereinthe starting sequence is modified to yield derivative engineered miRNAprecursors that are processed to engineered mature miRNAs designed tosilence a specific target gene or genes other than the endogenoustarget(s) of the mature miRNAs natively processed from the “8-miR”cluster. Designing an artificial or engineered miRNA sequence can be assimple as substituting sequence that is complementary to the intendedtarget for nucleotides in the miRNA stem region of the miRNA precursor,as demonstrated by Zeng et al. (2002) Mol. Cell, 9:1327-1333. Onenon-limiting example of a general method for determining nucleotidechanges in the native miRNA sequence to produce the engineered miRNAprecursor includes the following steps:

-   -   (a) Selecting a unique target sequence of at least 18        nucleotides specific to the target gene, e.g., by using sequence        alignment tools such as BLAST (see, for example, Altschul et        al. (1990) J. Mol. Biol., 215:403-410; Altschul et al. (1997)        Nucleic Acids Res., 25:3389-3402), for example, of both maize        cDNA and genomic DNA databases, to identify target transcript        orthologues and any potential matches to unrelated genes,        thereby avoiding unintentional silencing of non-target        sequences.    -   (b) Analyzing the target gene for undesirable sequences (e.g.,        matches to sequences from non-target species), and score each        potential 19-mer segment for GC content, Reynolds score (see        Reynolds et al. (2004) Nature Biotechnol., 22:326-330), and        functional asymmetry characterized by a negative difference in        free energy (“ΔΔG”) (see Khvorova et al. (2003) Cell,        115:209-216). Preferably 19-mers are selected that have all or        most of the following characteristics: (1) a Reynolds        score >4, (2) a GC content between about 40% to about 60%, (3) a        negative AAG, (4) a terminal adenosine, (5) lack of a        consecutive run of 4 or more of the same nucleotide; (6) a        location near the 3′ terminus of the target gene; (7) minimal        differences from the miRNA precursor transcript. Preferably        multiple (3 or more) 19-mers are selected for testing. Positions        at every third nucleotide in an siRNA have been reported to be        especially important in influencing RNAi efficacy and an        algorithm, “siExplorer” is publicly available at        rna.chem.t.u-tokyo.acjp/siexplorer.htm (see Katoh and        Suzuki (2007) Nucleic Acids Res., 10.1093/nar/gkl1120).    -   (c) Determining the reverse complement of the selected 19-mers        to use in making a modified mature miRNA. The additional        nucleotide at position 20 is preferably matched to the selected        target sequence, and the nucleotide at position 21 is preferably        chosen to either be unpaired to prevent spreading of silencing        on the target transcript or paired to the target sequence to        promote spreading of silencing on the target transcript.    -   (d) Testing the engineered miRNA precursor, for desirable        characteristics, such as in planta stability, or efficacy in        controlling the invertebrate pest. For example, a recombinant        DNA construct containing the engineered miRNA precursor is        expressed in plants under either a constitutive (e.g., CaMV 35S)        or tissue-specific (e.g., root) promoter and in planta stability        of the precursor is measured. In another example, the engineered        miRNA precursor can be tested in a bioassay for larval mortality        as a proxy measurement of target gene silencing efficacy; see,        for example, the Western corn rootworm (Diabrotica virgifera        virgifera LeConte) larval bioassay described in detail in        International Patent Application Publication WO2005/110068 A2        and U. S. Patent Application Publication US 2006/0021087 A1,        incorporated by reference. In yet another example, the        engineered miRNA precursor can be tested in a soybean cyst        nematode bioassay such as that described in detail by Steeves et        al. (2006) Funct. Plant Biol., 33:991-999, wherein cysts per        plant, cysts per gram root, eggs per plant, eggs per gram root,        and eggs per cyst are measured.    -   and (e) Cloning the most effective engineered miRNA precursor        into a construct for stable transformation of a plant, e.g.,        maize (see the sections under the headings “Making and Using        Recombinant DNA Constructs” and “Making and Using Transgenic        Plant Cells and Transgenic Plants”).

In a non-limiting example of the engineered miRNA approach, an“8-miR”sequence (SEQ ID NO. 1) was engineered to be processed to novelmature miRNAs designed to silence a Diabrotica virgifera vacuolar ATPase(vATPase, SEQ ID NO. 19). The mature miRNA sequences were designedtaking into account what is currently known about animal miRNAprocessing, see, for example, Schwarz et al. (2003) Cell, 115:199-208,Khvorova et al. (2003) Cell, 115:209-216, and Reynolds et al. (2004).Nature Biotechnol., 22:326-330. Perfect or near perfect matches to thetarget sequence were selected for minimal off-target effects and maximumpredicted efficacy. Each engineered mature miRNA sequence was designedto replace the corresponding endogenous mature miRNA, while preservingthe predicted secondary structure of the precursor and the “8-miR”cluster. Table 2 lists each of the eight selected target sequences, itslocation within SEQ ID NO. 19, relevant properties of each targetsequence, and the engineered miRNA sequence designed to silence thetarget sequence. The secondary structure (i.e., fold-back structure) ofeach native miRNA precursor was maintained in the correspondingengineered miRNA, which is depicted in FIG. 3.

The eight engineered miRNA sequences were substituted into the scaffold(native) “8-miR” cluster to yield a single engineered “8mirvATPase”sequence (SEQ ID NO. 44). Due to the method of construction, fivevariant 8mirvATPase sequences (each slightly different from SEQ ID NO.44) were obtained, including variants “8mirvATPase-11” (SEQ ID NO. 45)and “8mirvATPase-16” (SEQ ID NO. 46). RNA of each of the five versionsof 8mirvATPase was expressed from its corresponding plasmid and testedin a Western corn rootworm (WCR, Diabrotica virgifera) larval dietbioassay (described in U. S. Patent Application Publication US2006/0021087 A1, incorporated by reference). All five RNAs showed somemortality and stunting against WCR larvae. RNA from constructs pMON97871(containing SEQ ID NO. 45) and pMON97872 (containing SEQ ID NO. 46)caused significant WCR larval mortality of 64% and 77%, respectively,and were engineered for transcription in maize plants.

To test in planta stability of the engineered miRNA precursortranscripts, plasmid pMON97878 (including SEQ ID NO. 46 under thecontrol of the CaMv35S promoter) was made and infiltrated into tobacco(Nicotiana benthamiana) plants. After three days, total RNA wasextracted from three separate infiltrated plants and assayed by Northernblots using a digoxygenin-labelled RNA probe complementary to“8mirvATPase-16”. The Northern blot (FIG. 4) showed that“8mirvATPase-16” (SEQ ID NO. 46) RNA was present in tobacco plants asboth full-length “8mirvATPase-16” transcript and as degraded RNA,demonstrating that “8mirvATPase-16” RNA is more stable in plants than isthe corresponding double-stranded RNA produced from an inverted repeat(i.e., sense adjacent to anti-sense of the same target gene), which wasfound to be entirely cleaved to small RNAs in planta (data not shown).

To assess efficacy at conferring on the plant resistance to theinvertebrate pest, plasmids pMON97875, containing “8mirvATPase-11” (SEQID NO. 45), and pMON97876, containing “8mirvATPase-16” (SEQ ID NO. 46),were cloned using a maize binary plasmid. These plasmids weretransformed into maize. The resulting plants are expected to displayresistance to damage by Western corn rootworm.

TABLE 2 Start and end base Anti- DNA encoding of Sense sense Scaffoldengineered SEQ ID Reynolds' 5′ 5′ miRNA miRNA engineered miRNA Targetsequence NO. 19 GC % score mfe* mfe* ΔΔG precursor precursor precursorGACTGGCTTGGATCATATT 1423-1441 42.1 7 −9.2 −4.7 −4.5 mir309 SEQ ID NO. 28mir309vATPase1423 (SEQ ID NO. 20) (SEQ ID (SEQ ID NO. 36) NO. 11)GAGCATTGGACGACTTTTA 1454-1472 42.1 8 −10.6 −4.7 −5.9 mir3 SEQ ID NO. 29mir3vATPase1454 (SEQ ID NO. 21) (SEQ ID (SEQ ID NO. 37) NO. 12)TGTGCAGCTGGTAGGTAAA 1554-1572 47.4 7 −10.6 −6.0 −4.6 mir286 SEQ ID NO.30 mir286vATPase1554 (SEQ ID NO. 22) (SEQ ID (SEQ ID NO. 38) NO. 13)TGGCAGAAACGGACAAAAT 1580-1598 42.1 7 −11.5 −4.4 −7.1 mir4 SEQ ID NO. 31mir4vATPase1580 (SEQ ID NO. 23) (SEQ ID (SEQ ID NO. 39) NO. 14)TGCCAGGCTTCTTAAAGAA 1611-1629 42.1 8 −11.5 −6.7 −4.8 mir5 SEQ ID NO. 32mir5vATPase1611 (SEQ ID NO. 24) (SEQ ID (SEQ ID NO. 40) NO. 15)GCAGGCCTTCAGAAACTTA 1905-1923 47.4 8 −11.2 −6.8 −4.4 mir6-1 SEQ ID NO.33 mir6.1vATPase1905 (SEQ ID NO. 25) (SEQ ID (SEQ ID NO. 41) NO. 16)TGGCCGTACTAAAGATAGT 2046-2064 42.1 7 −12.6 −6.8 −5.8 mir6-2 SEQ ID NO.34 mir6.2vATPase2046 (SEQ ID NO. 26) (SEQ ID (SEQ ID NO. 42) NO. 17)GAGGGTACAGTGCTTATTA 2153-2171 42.1 8 −11.2 −5.0 −6.2 mir6-3 SEQ ID NO.35 mir6.3vATPase2153 (SEQ ID NO. 27) (SEQ ID (SEQ ID NO. 43) NO. 18)*mfe, minimum folding energy, a measure of the strongest pairinginteracting between two sequences measured as ΔG

Example 2

This example describes non-limiting embodiments including novelinvertebrate miRNA precursors. More particularly, this example describesnovel mature miRNA sequences and their corresponding miRNA precursorsequences and foldback structures, identified from soybean cyst nematode(SCN, Heterodera glycines). The nematode miRNA precursors are useful inmaking recombinant DNA constructs useful in making the non-naturaltransgenic plant cells, plants, and seeds of this invention, especiallynon-natural transgenic soybean plants having resistance to SCN and othernematode or invertebrate pests. Ingestion of nematode-specific miRNAs(whether as an exogenously expressed native sequence or as an engineeredmiRNA) by nematodes (e.g., SCN) is envisioned as a method of controllingnematode and other invertebrate pests.

A library of small RNAs from soybean cyst nematode (Heterodera glycine,SCN) was constructed by standard procedures similar to those describedin Aravin and Tuschl (2005) FEBS Letts., 579:5830-5840 and Ambros andLee (2004) Methods Mol. Bio., 265:131-158. In brief, total RNA wasisolated from SCN by the Trizol (Invitrogen) method. SCN RNA wasfractionated on a polyacrylamide gel and small RNAs (about 18 to about26 nucleotides) were eluted from the gel. Adaptors were ligated to the5′ and 3′ ends of the small RNAs and the resulting ligation mixture wasamplified by polymerase chain reaction (PCR). The PCR product wasligated into pCR2.1-TOPO vector (Invitrogen). The ligation mixture wastransformed into E. coli. One hundred ninety-two resulting transformedcolonies were grown and plasmid DNA was prepared from each colony. Apartial DNA sequence was determined for each plasmid to determine thenature of the small RNA inserted into the vector.

Two libraries were sequenced using conventional methods and 192 rawsequences were obtained. Eighty-nine unique small RNAs of about 18 toabout 26 nucleotides in length were retrieved and analyzed for newmiRNAs. New miRNAs were predicted by first folding the secondarystructure using the RNAfold program in the Vienna package as describedby Hofacker et al. (1994) Monatsh. f Chemie, 125:167-188. The structuresthus predicted were filtered based on characteristics of validated miRNAprecursors modified from those derived by Jones-Rhoades et al. (2006)Annu. Rev. Plant. Biol., 57:19-53. Finally the prediction result wasmanually inspected. SCN homologues to four published miRNA families wereidentified and are listed in Table 3. Additionally, from 52 SCN genomicloci, 4 novel SCN miRNAs were predicted and given the trivialidentifiers “SCN15” (GUCAGCCGAUCCUAAGGCACC, SEQ ID NO. 53), “SCN25”(UGGUGCGUGGACUAGUGGUGAG, SEQ ID NO. 54), “SCN30”(UGAAAGACAUGGGUAGUAUGAGACG, SEQ ID NO. 55), and “SCN31”(CACCUAUACUCCACCGUCAUUGG, SEQ ID NO. 56). The mature SCN miRNAs andtheir corresponding miRNA precursors are listed in Table 4. Non-limitingexamples of fold-back structures (i.e., secondary structures ofinvertebrate miRNA precursors, each including at least one stem-loopthat is processed to a mature miRNA, wherein the stem-loop includes astem region and a loop region) are depicted in FIG. 5.

TABLE 3 miRNA family SCN sequence SEQ ID NO. miR-8UAAUACUGUCAGGUAAAGAUGUC 47 miR-71 UGAAAGACAUGGGUAGUAUGAGACG 48 miR_86UAAGUGAAUUCUUUGCCACAGUCU 49 miR-100 AACCCGUAGAUCCGAACUAGUC 50 miR-100AACCCGUAGAUCCGAACUAGUCU 51 miR-100 AACCCGUAGAUCCGAACUUGUG 52

TABLE 4 Nucleotide Nucleotide position of position of mature mature Pre-pre-miRNA SCN miRNA miRNA in miRNA in genomic mature SEQ ID pre-miRNASEQ ID locus miRNA NO. start end NO start end SCN genomic locus 15 53 5979 57 181 270 HG3_LIB5513-477-A1-M1-H4 25 54 43 64 58 197 271HG3_34113.C1 25 54 43 64 58 197 271 HG3_29652.C1 25 54 43 64 58 197 271HG3_28454.C1 25 54 43 64 59 197 271 HG3_18268.C1 25 54 43 64 58 197 271HG3_34533.C1 25 54 43 64 58 172 246 HG3_19260.C1 25 54 43 64 58 103 177HG3_25275.C1 25 54 43 64 60 103 177 HG3_12307.C1 25 54 43 64 58 197 271HG3_25338.C1 25 54 43 64 58 197 271 HG3_10253.C1 25 54 43 64 58 154 228HG3_29286.C1 25 54 43 64 58 197 271 HG3_254.C1 25 54 43 64 58 156 230HG3_8212.C1 25 54 43 64 59 197 271 HG3_18844.C1 25 54 43 64 61 63 137HG3_1908.C2 25 54 43 64 58 197 271 HG3_33191.C1 25 54 43 64 59 197 271HG3_LIB5513-515-A1-P1-C7 25 54 43 64 58 197 271 HG3_LIB5519-246-A1-M1-A225 54 43 64 59 35 109 HG3_LIB5520-450-A1-P1-D6 25 54 43 64 58 197 271HG3_LIB5519-364-A1-M1-C2 25 54 43 64 58 197 271HG3_LIB5513-505-A2-M1-E11 25 54 43 64 62 197 271HG3_LIB5513-708-A1-M1-E9 25 54 43 64 59 197 271HG3_LIB5513-353-A1-P1-B11 25 54 43 64 59 197 271HG3_LIB5513-678-A1-M1-E6 25 54 43 64 63 76 150 HG3_LIB5513-103-A1-M1-G425 54 43 64 59 197 271 HG3_LIB5519-495-A1-M1-G5 25 54 43 64 59 48 122HG3_LIB5514-043-A1-P1-D11 25 54 43 64 62 197 271HG3_LIB5513-373-A1-M1-H2 25 54 43 64 58 197 271 HG3_LIB5513-801-A1-P1-D525 54 43 64 58 197 271 HG3_LIB5519-564-A1-P1-C9 25 54 43 64 59 35 109HG3_LIB5519-028-A1-P1-B10 25 54 43 64 58 197 271HG3_LIB5519-120-A1-P1-F12 25 54 43 64 64 197 271HG3_LIB5519-507-A1-M1-F3 25 54 43 64 59 197 271 HG3_LIB5519-231-A1-M1-G225 54 43 64 59 197 271 HG3_LIB5519-518-A1-M1-B2 25 54 43 64 58 197 271HG3_LIB5513-224-A1-P1-E3 25 54 43 64 58 164 238 HG3_LIB5513-691-A1-M1-G525 54 43 64 58 197 271 HG3_LIB5519-295-A1-M1-G11 25 54 43 64 58 197 271HG3_LIB5513-052-A1-M1-F7 25 54 43 64 58 197 271HG3_LIB5519-450-A1-P1-F10 30 55 10 34 65 228 319 HG3_1898.C5 31 56 65 8766 696 793 HG3_25240.C1 31 56 65 87 67 696 793 HG3_23769.C1 31 56 65 8766 690 787 HG3_13335.C1 31 56 65 87 66 699 796 HG3_21499.C1 31 56 64 8668 151 247 HG3_LIB5513-288-A1-M1-G12 31 56 64 86 68 49 145HG3_LIB5513-004-A1-M1-C6 31 56 10 32 69 133 230 HG3_LIB5520-318-A1-P1-D731 56 64 86 68 140 236 HG3_LIB5519-222-A1-P1-D9 31 56 64 86 68 174 270HG3_LIB5520-295-A1-M1-E8 31 56 64 86 68 138 234 HG3_LIB5519-532-A1-M1-E2

Example 3

This example describes another non-limiting embodiment of a recombinantDNA construct that is transcribable in a plant cell to a recombinantmiRNA precursor, preferably conferring on the plant resistance to aninvertebrate pest. More particularly, this embodiment describes arecombinant DNA construct that includes sequence derived from multiplemiRNA precursors, in this case nine invertebrate miRNA precursors thathave homology to Western corn rootworm (WCR, Diabrotica virgifera) miRNAprecursors.

From a library of small RNAs from Western corn rootworm was obtainednine clones with homology to known miRNAs; these putative WCR miRNAs aregiven in Table 5.

TABLE 5 SEQ Small miRNA ID Size RNA ID family NO. cloned WCR miRNA (nt)2997059 bta-miR-7 70 TGGAAGACTAGTGATTTTGTTGTT 24 1167198 dme-miR- 71TCTTTGGTTATCTAGCTGTATGA 21 9a 2999692 dme-miR- 72 TCAGTCTTTTTCTCTCTCCTA22 14 3000551 dme-miR- 73 GGCAAGATGTCGGCATAGCTG 22 31a 3000395 sme-miR-74 TGAAAGACATGGGTAGTGAGAT 22 71c 3000406 aga-miR- 75AATTGCACTTGTCCCGGCCTGC 22 92b 2998018 dme-miR- 76 TCAGGTACCTGAAGTAGCGCGC22 275 2833118 dme-miR- 77 TGACTAGATCCACACTCATTAA 23 279 3007470dme-miR- 78 ATTGTACTTCATCAGGTGCTCT 21 305

For each of the nine microRNAs, invertebrate pre-miRNA homologues wereidentified from MiRbase (eight from the fruit fly Drosophilamelanogaster and one from the nematode Caenorhabdites elegans). Theselected homologous pre-miRNA sequences (each including an additional 10nucleotides upstream and downstream of the pre-miRNA, as indicated bythe “+10” in the sequence name) were: “dme-mir-7+10”(GTCCTCCTGGGAGTGCATTCCGTATGGAAGACTAGTGATTTTGTTGTTTGGTCTTTGGTAATAACAATAAATCCCTTGTCTTCTTACGGCGTGCATTTGTGCTCTTCA, SEQ ID NO. 79),“dme-mir-9a+10”(TATACAGGGTGCTATGTTGTCTTTGGTTATCTAGCTGTATGAGTGATAAATAACGTCATAAAGCTAGCTTACCGAAGTTAATATTAGCGTCTGCCCAG, SEQ ID NO. 80), “dme-mir-14+10”(CTGCAACCTATGTGGGAGCGAGACGGGGACTCACTGTGCTTATTAAATAGTCAGTCTTTTTCTCTCTCCTATACAAATTGCGG, SEQ ID NO. 81), “dme-mir-31a+10”(CGCTGACTGTTCCATTGAACAACTGACTAGATGCAGCATAGCGCTCTTCAAAATCGCTTTTCAACGTCAGCTATGCCGACATCTTGCCAATTTACCAACGGAGTTGATATAC, SEQ ID NO. 82),“Cel-miR-71+10”(CACAGAGGTTGTCTGCTCTGAACGATGAAAGACATGGGTAGTGAGACGTCGGAGCCTCGTCGTATCACTATTCTGTTTTTCGCCGTCGGGATCGTGACCTGGAAGCTGTAAACT, SEQ ID NO.83), “dme-mir92a+10”(GCCGAATATAAATATGAATTTCCCGTAGGACGGGAAGGTGTCAACGTTTTGCATTTCGAATAAACATTGCACTTGTCCCGGCCTATGGGCGGTTTGTAATAAACAACTAAAATCT, SEQ ID NO.84), “dme-miR275+10”(TTCCCCCGACTGTAAAGTCTCCTACCTTGCGCGCTAATCAGTGACCGGGGCTGGTTTTTTATATACAGTCAGGTACCTGAAGTAGCGCGCGTGGTGGCAGACATATATCTCCATCTTC, SEQ ID NO.85), “dme-miR279+10”(AGCTGGAATTGGAATTCATACTACTGTTTTTAGTGGGTGGGGGTCCAGTGTTTCACATTGATTTTCTTAGTATTTGTGACTAGATCCACACTCATTAATAACGGTAGTTCAATCATCA AG, SEQ IDNO. 86), and “dme-miR305+10”(AACTGTCTCCCATGTCTATTGTACTTCATCAGGTGCTCTGGTGTGTCTCGTAACCCGGCACATGTTGAAGTACACTCAATATGAGGCGATTTG, SEQ ID NO. 87).

The nine pre-miRNAs (including the extra “+10” nucleotides) were joinedhead-to-tail to yield a 951-nucleotide sequence,“miR-7+9+14+31+71+92+275+279+305”(GTCCTCCTGGGAGTGCATTCCGTATGGAAGACTAGTGATTTTGTTGTTTGGTCTTTGGTAATAACAATAAATCCCTTGTCTTCTTACGGCGTGCATTTGTGCTCTTCATATACAGGGTGCTATGTTGTCTTTGGTTATCTAGCTGTATGAGTGATAAATAACGTCATAAAGCTAGCTTACCGAAGTTAATATTAGCGTCTGCCCAGCTGCAACCTATGTGGGAGCGAGACGGGGACTCACTGTGCTTATTAAATAGTCAGTCTTTTTCTCTCTCCTATACAAATTGCGGCGCTGACTGTTCCATTGAACAACTGACTAGATGCAGCATAGCGCTCTTCAAAATCGCTTTTCAACGTCAGCTATGCCGACATCTTGCCAATTTACCAACGGAGTTGATATACCACAGAGGTTGTCTGCTCTGAACGATGAAAGACATGGGTAGTGAGACGTCGGAGCCTCGTCGTATCACTATTCTGTTTTTCGCCGTCGGGATCGTGACCTGGAAGCTGTAAACTGCCGAATATAAATATGAATTTCCCGTAGGACGGGAAGGTGTCAACGTTTTGCATTTCGAATAAACATTGCACTTGTCCCGGCCTATGGGCGGTTTGTAATAAACAACTAAAATCTTTCCCCCGACTGTAAAGTCTCCTACCTTGCGCGCTAATCAGTGACCGGGGCTGGTTTTTTATATACAGTCAGGTACCTGAAGTAGCGCGCGTGGTGGCAGACATATATCTCCATCTTCAGCTGGAATTGGAATTCATACTACTGTTTTTAGTGGGTGGGGGTCCAGTGTTTCACATTGATTTTCTTAGTATTTGTGACTAGATCCACACTCATTAATAACGGTAGTTCAATCATCAAGAACTGTCTCCCATGTCTATTGTACTTCATCAGGTGCTCTGGTGTGTCTCGTAACCCGGCACATGTTGAAGTACACT CAATATG, SEQID NO. 88). This was synthesized by PCR using overlapping 50-meroligonucleotides. The sequence was cloned into pCR4-TOPO vector(Invitrogen), yielding plasmid pMON97886, from which T7 polymerase wasused to generate RNA.

The resulting RNA corresponding to SEQ ID NO. 88 is tested in a Westerncorn rootworm (WCR, Diabrotica virgifera) larval diet bioassay(described in U. S. Patent Application Publication US 2006/0021087 A1,incorporated by reference), and mortality and stunting against WCRlarvae is measured.

Example 4

This example describes another non-limiting embodiment of a recombinantDNA construct that is transcribable in a plant cell to a recombinantmiRNA precursor, preferably conferring on the plant resistance to aninvertebrate pest. More particularly, this embodiment describes arecombinant DNA construct that includes artificial miRNA precursorsequences derived from soybean cyst nematode (Heterodera glycine, SCN)miRNA sequences engineered to suppress an invertebrate target gene.

When expressed as dsRNA in soybean, major sperm protein1 (MSP1) has beenreported to cause significant mortality in H. glycines; see Steeves etal. (2006) Funct. Plant Biol., 33:991-999. A 21-nucleotide sequence,TCTTGAGACTGTCCTGTATTA (SEQ ID NO. 89), from H. glycines MSP1 (SEQ ID NO.90) was selected as the target sequence, based on functional asymmetryand efficacy predictors as described by Reynolds et al. (2004) NatureBiotechnol., 22:326-330 and Khvorova et al. (2003) Cell, 115:209-216.The “SCN15” pre-miRNA sequence (SEQ ID NO. 57) that produces the “SCN15”mature miRNA (SEQ ID NO. 53) (see Example 2) was modified by replacingthe nucleotides of the “SCN15” mature miRNA with the engineered maturemiRNA “SCN15-miRMSP1” (SEQ ID NO. 91), and the complementary region ofthe foldback was changed to maintain the original paired and unpairedbases, yielding the engineered pre-miRNA sequence “SCN15-MIRMSP1” (SEQID NO. 92); see FIG. 6A. The synthetic pre-miRNA “SCN15-MIRMSP1” (SEQ IDNO. 92) is cloned into a binary expression vector with a constitutive orroot-specific promoter, transformed into soybean, and assayed forefficacy at protecting against SCN, e.g., with the bioassay described bySteeves et al. (2006) Funct. Plant Biol., 33:991-999).

Example 5

This example describes another non-limiting embodiment of a recombinantDNA construct that is transcribable in a plant cell to a recombinantmiRNA precursor, preferably conferring on the plant resistance to aninvertebrate pest. More particularly, this embodiment describes arecombinant DNA construct that includes artificial miRNA precursorsequences derived from soybean cyst nematode (Heterodera glycine, SCN)miRNA sequences engineered to suppress an invertebrate target gene.

A 22-nucleotide sequence, TGGTGCGTGGACTAGTGGTGAG (SEQ ID NO. 93), fromH. glycines cgh-1 (SEQ ID NO. 94) was selected as the target sequence,based on functional asymmetry and efficacy predictors as described byReynolds et al (2004) Nature Biotechnol., 22:326-330 and Khvorova et al.(2003) Cell, 115:209-216. An “SCN25” pre-miRNA sequence (SEQ ID NO. 58)that produces the “SCN25” mature miRNA (SEQ ID NO. 54) (see Example 2)was modified by replacing the nucleotides of the “SCN25” mature miRNAwith the engineered mature miRNA “SCN25-miRcgh1” (SEQ ID NO. 95), andthe complementary region of the foldback was changed to maintain theoriginal paired and unpaired bases, yielding the engineered pre-miRNAsequence “SCN25-MIRcgh1” (SEQ ID NO. 96); see FIG. 6B. The syntheticpre-miRNA “SCN25-MIRcgh1” (SEQ ID NO. 96) is cloned into a binaryexpression vector with a constitutive or root-specific promoter,transformed into soybean, and assayed for efficacy at protecting againstSCN, e.g., with the bioassay described by Steeves et al. (2006) Funct.Plant Biol., 33:991-999).

Example 6

In many embodiments of this invention, the recombinant DNA constructfurther includes one or more elements selected from: (a) a promoterfunctional in a plant cell; (b) a transgene transcription unit; (c) agene suppression element; and (d) a transcription regulatory/transcriptstabilizing element. This example further illustrates non-limitingembodiments of gene suppression elements. Gene suppression elements caninclude, but are not limited to, the invertebrate mature miRNAs andmiRNA precursors that are aspects of this invention.

FIG. 7A schematically depicts non-limiting examples of recombinant DNAconstructs that illustrate arrangement of components of the construct.In these non-limiting examples, the constructs include at least onefirst gene suppression element (“GSE” or “GSE1” ) for suppressing atleast one first target gene, wherein the first gene suppression elementis embedded in an intron flanked on one or on both sides bynon-protein-coding DNA. These constructs utilize an intron (in manyembodiments, an intron derived from a 5′ untranslated region or anexpression-enhancing intron is preferred) to deliver a gene suppressionelement without requiring the presence of any protein-coding exons(coding sequence). The constructs can optionally include at least onesecond gene suppression element (“GSE2”) for suppressing at least onesecond target gene, at least one gene expression element (“GEE”) forexpressing at least one gene of interest (which can be coding ornon-coding sequence or both), or both. In embodiments containing anoptional gene expression element, the gene expression element can belocated outside of (e.g., adjacent to) the intron. In some embodiments,the intron containing the first gene suppression element is 3′ to aterminator.

To more clearly differentiate recombinant DNA constructs of theinvention (containing at least one gene suppression element embeddedwithin a single intron flanked on one or on both sides bynon-protein-coding DNA) from the prior art, FIG. 7B schematicallydepicts examples of prior art recombinant DNA constructs. Theseconstructs can contain a gene suppression element that is locatedadjacent to an intron flanked by protein-coding sequence, or between twodiscrete introns (wherein the gene suppression element is not embeddedin either of the two discrete introns), or can include a gene expressionelement including a gene suppression element embedded within an intronwhich is flanked by multiple exons (e.g., exons including the codingsequence of a protein).

FIG. 8 depicts various non-limiting examples of gene suppressionelements useful in the recombinant DNA constructs of the invention.Where drawn as a single strand (FIGS. 8A through 8E), these areconventionally depicted in 5′ to 3′ (left to right) transcriptionaldirection; the arrows indicate anti-sense sequence (arrowhead pointingto the left), or sense sequence (arrowhead pointing to the right). Thesegene suppression elements can include: DNA that includes at least oneanti-sense DNA segment that is anti-sense to at least one segment of atleast one first target gene, or DNA that includes multiple copies of atleast one anti-sense DNA segment that is anti-sense to at least onesegment of at least one first target gene (FIG. 8A); DNA that includesat least one sense DNA segment that is at least one segment of at leastone first target gene, or DNA that includes multiple copies of at leastone sense DNA segment that is at least one segment of at least one firsttarget gene (FIG. 8B); DNA that transcribes to RNA for suppressing atleast one first target gene by forming double-stranded RNA and includesat least one anti-sense DNA segment that is anti-sense to at least onesegment of at least one first target gene and at least one sense DNAsegment that is at least one segment of at least one first target gene(FIG. 8C); DNA that transcribes to RNA for suppressing at least onefirst target gene by forming a single double-stranded RNA and includesmultiple serial anti-sense DNA segments that are anti-sense to at leastone segment of at least one first target gene and multiple serial senseDNA segments that are at least one segment of at least one first targetgene (FIG. 8D); DNA that transcribes to RNA for suppressing at least onefirst target gene by forming multiple double strands of RNA and includesmultiple anti-sense DNA segments that are anti-sense to at least onesegment of at least one first target gene and multiple sense DNAsegments that are at least one segment of at least one first targetgene, and wherein the multiple anti-sense DNA segments and the multiplesense DNA segments are arranged in a series of inverted repeats (FIG.8E); and DNA that includes nucleotides derived from a miRNA (including,but not limited to, nucleotides derived from the invertebrate maturemiRNAs and miRNA precursors of this invention), or DNA that includesnucleotides of a siRNA (FIG. 8F).

FIG. 8F depicts various non-limiting arrangements of double-stranded RNAthat can be transcribed from embodiments of the gene suppressionelements useful in the recombinant DNA constructs of the invention. Whensuch double-stranded RNA is formed, it can suppress one or more targetgenes, and can form a single double-stranded RNA or multiple doublestrands of RNA, or a single double-stranded RNA “stem” or multiple“stems”. Where multiple double-stranded RNA “stems” are formed, they canbe arranged in “hammerheads” or “cloverleaf” arrangements. In someembodiments, the double-stranded stems can form a “pseudoknot”arrangement (e.g., where spacer or loop RNA of one double-stranded stemforms part of a second double-stranded stem); see, for example,depictions of pseudoknot architectures in Staple and Butcher (2005) PLoSBiol., 3(6):e213. Spacer DNA (located between or adjacent to dsRNAregions) is optional but commonly included and generally includes DNAthat does not correspond to the target gene (although in someembodiments can include sense or anti-sense DNA of the target gene).Spacer DNA can include sequence that transcribes to single-stranded RNAor to at least partially double-stranded RNA (such as in a “kissingstem-loop” arrangement), or to an RNA that assumes a secondary structureor three-dimensional configuration (e.g., a large loop of antisensesequence of the target gene or an aptamer) that confers on thetranscript an additional desired characteristic, such as increasedstability, increased half-life in vivo, or cell or tissue specificity.

All of the materials and methods disclosed and claimed herein can bemade and used without undue experimentation as instructed by the abovedisclosure. Although the materials and methods of this invention havebeen described in terms of preferred embodiments and illustrativeexamples, it will be apparent to those of skill in the art thatvariations can be applied to the materials and methods described hereinwithout departing from the concept, spirit and scope of the invention.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

1. A non-natural plant having resistance to an invertebrate pest thatingests RNA from said plant, wherein said non-natural plant comprises atransgenic plant cell having in its genome a recombinant DNA constructthat is transcribed in said transgenic plant cell to a recombinant miRNAprecursor; and said recombinant miRNA precursor comprises a singlestrand of RNA that folds into the secondary structure of an invertebratemiRNA precursor and that comprises at least one stem-loop that isprocessed to a mature miRNA; and said mature miRNA suppresses expressionof at least one target gene of said invertebrate pest or of a symbiontassociated with said invertebrate pest, thereby conferring on saidnon-natural plant resistance to said invertebrate pest.
 2. Thenon-natural plant of claim 1, wherein said at least one stem-loop thatis processed to a mature miRNA comprises a stem region and a loopregion, wherein said stem region comprises a first segment and a secondsegment, said first segment comprises at least 19 contiguous nucleotidesfor silencing a messenger RNA encoding said target gene, said secondsegment comprises at least 19 contiguous nucleotides, said loop regionis located between said first and second segments, and said first andsecond segments hybridize to form partially double stranded RNA, whereinat least one nucleotide of said first segment is unpaired.
 3. Thenon-natural plant of claim 3, wherein said at least 19 contiguousnucleotides for silencing a messenger RNA have at least 70%complementarity to a segment of equivalent length in said messenger RNA.4. The non-natural plant of claim 3, wherein said loop region comprisesa native loop sequence of said invertebrate miRNA precursor.
 5. Thenon-natural plant of claim 1, wherein said single strand of RNAcomprises a single stem-loop that is processed to a mature miRNA.
 6. Thenon-natural plant of claim 1, wherein said single strand of RNAcomprises multiple stem-loops that are processed to mature miRNAs. 7.The non-natural plant of claim 1, wherein said invertebrate pest is atleast one selected from the group consisting of insects, arachnids,nematodes, molluscs, and annelids.
 8. The non-natural plant of claim 1,wherein said at least one target gene is (a) a single target gene, or(b) multiple target genes.
 9. The non-natural plant of claim 1, whereinsaid at least one target gene is an endogenous target of an invertebratemiRNA natively expressed from said invertebrate miRNA precursor.
 10. Thenon-natural plant of claim 1, wherein said at least one target gene isother than an endogenous target of an invertebrate miRNA nativelyexpressed from said invertebrate miRNA precursor.
 11. The non-naturalplant of claim 1, wherein said at least one target gene is a target geneof a symbiont associated with said invertebrate pest.
 12. Thenon-natural plant of claim 1, wherein (a) said non-natural plant ismaize and said invertebrate pest is a Diabrotica species; (b) saidnon-natural plant is soybean and said invertebrate pest is a soybeancyst nematode; (c) said non-natural plant is a grape and saidinvertebrate pest is a grape phylloxera or a glassy-winged sharpshooter(Homalodisca coagulata); or (d) said non-natural plant is an apple andsaid invertebrate pest is a woolly apple aphid
 13. The non-natural plantof claim 1, wherein said recombinant DNA construct further comprises oneor more elements selected from: (a) a promoter functional in a plantcell; (b) a transgene transcription unit; (c) a gene suppressionelement; and (d) a transcription regulatory/transcript stabilizingelement.
 14. The non-natural plant of claim 1, wherein said non-naturalplant is a transgenic plant.
 15. The non-natural plant of claim 1,wherein said non-natural plant further comprises non-transgenic tissue.16. The non-natural plant of claim 15, wherein said plant comprises anon-transgenic scion and a transgenic rootstock comprising saidtransgenic plant cell.
 17. A non-natural transgenic plant cell having inits genome a recombinant DNA construct that is transcribed in saidnon-natural transgenic plant cell to a recombinant miRNA precursor,wherein said recombinant miRNA precursor comprises a single strand ofRNA that folds into the secondary structure of an invertebrate miRNAprecursor and that comprises at least one stem-loop that is processed toa mature miRNA, and wherein said mature miRNA suppresses expression ofat least one target gene of an invertebrate or of a symbiont associatedwith said invertebrate.
 18. A non-natural transgenic plant grown fromthe non-natural transgenic plant cell of claim
 17. 19. Non-naturaltransgenic seed produced by the non-natural transgenic plant of claim18.
 20. A method of suppressing at least one target gene of aninvertebrate pest of a plant, comprising providing a plant comprisingthe non-natural transgenic plant cell of claim 17, wherein saidinvertebrate is said invertebrate pest, said recombinant DNA constructis transcribed in said non-natural transgenic plant cell to saidrecombinant miRNA precursor, and when said invertebrate pest ingestssaid recombinant miRNA precursor, said at least one target gene issuppressed.